An extended-release tablet of tacrolimus as once-daily dosing was fabricated using 3D printing technology. It was developed by combining two 3D-printing methods in parallel. Indeed, an optimized mixture of PVA, sorbitol, and magnesium stearate as a shell compartment was printed through a hot-melt extrusion (HME) nozzle while an HPMC gel mixture of the drug in the core compartment was printed by a pressure-assisted micro-syringe (PAM). A 3D-printed tablet with an infill of 90% was selected as an optimized formula upon the desired dissolution profile, releasing 86% of the drug at 12 h, similar to the commercial one. The weight variation, friability, hardness, assay, and content uniformity determination met USP requirements. A microbial evaluation showed that the 3D-printed tablet does not support microbial growth. SEM analysis showed smooth surfaces with multiple deposited layers. No peak interference appeared based on FTIR analysis. No decomposition of the polymer and drug was observed in the printing temperature, and no change in tacrolimus crystallinity was detected based on TGA and DSC analyses, respectively. The novel, sTable 3D-printed tablet, fabricated using controllable additive manufacturing, can quickly provide tailored dosing with specific kinetic release for personalized medicine at the point-of-care. Transplantation has been defined as the relocation of healthy tissues or organs to different parts of the recipient’s body, restoring the function of the faulty organ in the recipient. According to the recent WHO reports on approximately 80.4% of the world’s population, there have been roughly 12,981 solid organ transplants each year, comprising 31.7% kidney transplants, 20.1% liver transplants, and the remaining being related to heart, lung, pancreas and S, bowel transplantations [Immunosuppressant medication has been used to prevent/treat graft rejection and graft-versus-host diseases and has been considered to reduce tissue damage, which occurs in autoimmune or inflammatory reactions. The immunological response has been modulated via the following major drug classes: corticosteroids such as methylprednisolone and dexamethasone; cytotoxic suppressants such as chlorambucil, cyclophosphamide, mercaptopurine, and methotrexate; suppressant antibodies such as infliximab and basiliximab; mTOR-inhibitor derivatives of sirolimus such as everolimus; and finally, calcineurin inhibitors such as cyclosporine and tacrolimus .©, was commercially made for kidney and liver transplantations in 1994 [Tacrolimus is known as a macrolide inhibitor of calcineurin which was first isolated from bacterium streptomyces tsukubaensis in 1987 . Tacroli in 1994 ; however in 1994 . Evidenc in 1994 . TacroliControlled-release drug delivery systems for tacrolimus are reported on liposomal forms, nanoporous materials, electromechanics, and hydrogel-based and polymeric systems ,10,11,12©  and Advagraf© . These forms have shown better patient compliance with a reduced dosage of once per day, stable systemic absorption, and efficacy and safety comparable to common forms in patients with renal and liver transplants.Tacrolimus, in extended-release forms, has already become available under the brand names AstagrafDespite the improved efficacy in the extended-release form, concerns of toxicity or graft rejection related to immunosuppressant drugs’ pharmacologic and metabolic profiles must be considered for treatment. The variable bioavailability of tacrolimus, which is reported between 5% to 67% and is due to the first-pass effect, can put the patients at the risk of graft rejection. Furthermore, pharmacokinetic differences due to age, race, illnesses, and dissatisfaction with treatment lead to poor clinical consequences. For example, low absorption has been reported in children for tacrolimus FK506 due to increased removal by the liver and kidney. Furthermore, diabetic patients have shown reduced absorption for FK506, hence needing more drugs than non-diabetic patients; therefore, transplant recipients must undergo therapeutic dose monitoring to improve clinical efficacy and reduce toxicity caused by therapy .© , as the first 3D-printed drug approved by the FDA, is an example from such a method [Personalized medicine refers patients to particular medications according to their individual characteristics including their genetic profile, phenotypic response, and their pathology. New technologies should be introduced to produce standard dosage forms rapidly at the point-of-care. The production of personalized tablets has recently been facilitated by 3D-printing techniques and additive manufacturing ,17. Compa method .The most notable benefits of the approach are mentioned: (1) Stability of multiple drugs in a single tablet; (2) Producing an exact dose of each drug according to the pharmacokinetic profile; (3) Controlling the release kinetics of each drug in designed structure by balancing the dissolution profile .In pharmaceutics, hot-melt extrusion (HME) has been used to improve solubility, create a controlled release, and, in some cases, cover the harsh taste of the drugs . The HMEHere, we have formulated an extended-release tacrolimus tablet using extrusion-based 3D-printing techniques for the first time. The polymer mixture for the shell and drug-containing gel for the core compartment of printer inks were optimized to obtain the desired release profile. The 3D-printed tablet was then physiochemically characterized in terms of API and excipient interaction, decomposition, in vitro drug-release behavior, microorganism contamination, stability, and other USP requirements.Tacrolimus monohydrate was bought from sigma company . PVA filaments were purchased from Ultimaker company . Magnesium stearate and sorbitol were purchased from Merck CO. . Hypromellose was provided by Samsung Fine Chemicals Co. . All solvents were supplied from Merck CO.The pattern of the tablet form, with a round shape of about 12 mm in diameter and 5 mm in height, was designed with SolidWorks software . The dimension of the design was further modified based on experimental work. For the shell compartment, the various ratios of PVA filaments, magnesium stearate, and sorbitol were mixed and melted for 20 min to get an optimized ratio based on extrudability and printability in a hot-melt extruder. For the inner gel, different ratios of tacrolimus, ethanol, and HPMC were mixed. The best ratio based on extrudability and printability was chosen for printing using a second extruder, PAM. Finally, the filament and gel were introduced into the 3D printer .The tablet layout was uploaded by Repetier slicing software (version 2.2.4) and then printed with 3D Bio using two independent extruders. First, PVA filament was extruded and printed using working stepper motors which directed filaments into the hot-melt container equipped with a 0.4 mm nozzle. The printing process was conducted at 150–225 °C upon formulations at 10 mm/s speed, a bed temperature of 50 °C, and a layer height of 0.4 mm. In parallel, the second PAM conducted the drug-containing gel into the interval space of printed PVA shells in the core layer position.Tablet morphology was investigated using scanning electron microscopy (SEM)  at 10 kv and 54× to determine the dimensions of the printed layers of tablets. Before imaging, the sample was prepared on metallic stubs and gold plated in a vacuum chamber using JFC-1200 Fine Coater .−1 using infrared spectroscopy .The drug substance, excipient, and grinded powder from the printed tablets were mixed with KBR and then pressed separately to obtain disks. Each disk was placed in a sample position to scan the infrared spectrum in the range of 400 to 4000 cmA thermal analysis instrument  was utilized to investigate the thermal behavior of the material. Briefly, 5 mg of each sample was accurately weighed and placed in the Alu pan of the device. Then, samples were heated from 25 to 600 °C at a rate of 10 °C under a nitrogen medium to measure them in triplicate.All measurements were performed according to the USP 43 monograph for tacrolimus determination in the dosage form. To prepare the sample solution for assay, 20 tablets were mixed, accurately weighed into amounts of milled powder equivalent to the mean weight of tablets, transferred to a 250 mL measuring flask, dissolved, shaken, and made up to volume. For the content uniformity assessment, ten printed tablets were placed separately in 250 mL flasks to dissolve in the mobile phase in a procedure similar to the assay.For quantification, a standard stock solution of 200 μg/mL of tacrolimus was prepared and then diluted to standard solution in mobile phase with an equivalent concentration to the sample solution.For dissolution, samples were taken from 900 mL media (0.005% HPC in water with 0.50% SLS adjusted to pH 4.5) in time intervals of 0.5, 1, 2.5, 4.5, 6.5, 8.5, and 12 h using a dissolution tester (Erweka) in which the temperature and paddle rotation had been set at 37 °C and 100 rpm, respectively.To estimate the amount of tacrolimus in each sample, standard solutions were prepared from a common stock solution of assay after diluting it in dissolution media.A portion of each sample solution passed through a filter of 0.45 μM before the volume of 20 μl was injected into the HPLC.The chromatographic condition was adjusted to the mobile phase of 6% phosphoric acid; deionized water: methanol: acetonitrile with ratios of 0.1:36: 18: 46; column: 4.6 mm × 10 cm; 5 μm packing L1 ; detection wavelength of 210 nm; column temperature and flow rate were set at 50 °C and 1 mL/min, respectively. Chromatograms were taken for 25 min. The drug percentage was calculated according to the peak area of the tacrolimus in injected samples in comparison to that of the injected standards.The hardness and friability of tablets were measured using a hardness tester  and friability tester , respectively. Furthermore, weight variation was calculated using a balance  after weight measurement.E. coli, Salmonella species, Pseudomonas aeroginosa, Staphylococcus aureous, Candida albicans, and Aspergillus niger was examined according to US pharmacopeia <61> as described “Microbiological Examination of Nonsterile Products: Microbial Enumeration”.The streak plate method was used to determine the total microbial content using agar plates. Briefly, 1 gr of each sample was dispersed in sterile normal saline. An appropriate volume of sterile culture media of Mueller Hinton Agar and soybean casein digest agar were transferred into the plate and incubated for 24–48 h at 35 °C. Furthermore, to determine mold contamination, samples were cultured in sabro dextrose agar medium and incubated for 5–7 days at 25 °C. Finally, a differential diagnosis of The stability study of the 3D-printed tablets in a blister pack was carried out at 40 °C ± 2 °C/75 ± 5% RH for a duration of 36 months in a stability chamber, as recommended in ICH quality topics Q1.Due to concerns about the thermal instability of the tacrolimus during hot-melt extrusion, for the 3D printing of the tablet, PAM was also utilized in parallel to the hot-melt extruder. The layout of the tablet shell is shown in To fabricate the PVA filament for the shell part of the tablet, different ratios of polymer, plasticizer, and lubricant were mixed and melted to achieve extrudability and lower temperature printability, as shown in In addition, magnesium stearate was added to the formulation to improve extrudability. As shown, the addition of plasticizers of PEG35000 and PEG10000 did not result in printability and extrudability; however, the addition of sorbitol as the plasticizer at a considerable amount not only resolved the process of extrusion and printing, but also facilitated printing at a lower temperature. Different types of hot-melt extruded filaments upon optimization are represented in As shown in The sliced version by software and the image of the final 3D-printed tablet is shown in The morphology of the printed filament was investigated by SEM imaging B. The im−1. Furthermore, stretching vibrations of C-O at 1194.05 cm−1 for ester and C=O stretching at 1741.63, 1724, and 1694.29 cm−1 for ester and ketone bands were observed, respectively. Furthermore, the vibration of the O-H band at 3460 cm−1 indicated tacrolimus identification. Additionally, the 2925 and 2981 cm−1 bands are attributed to vibrations of symmetric CH2 and asymmetric C-H bands, respectively.The FTIR spectrum of tacrolimus, according to −1 for hydroxyl, 2925 cm−1 for vibrations of C-H and 1636 cm−1 band for C=C vibration, as well as 1111 cm−1 and 1384 cm−1 C-H for stretching vibrations of C-O bands  as shown in t) was around 19.5 min. The dissolution profile of the extended-release 3D-printed tablet of tacrolimus is shown in Additionally, the results of content uniformity of the tablets met the USP requirement, as the acceptance value was below 15  is within the limit, and the microbial contamination was not significant. Furthermore, a low level of fungi contamination was detected in the same sample . FurtherThe specifications for the 3D-printed tablets of ticagrelor at the end of their shelf life are demonstrated in One of the most critical objectives in developing pharmaceutical dosage forms is maintaining the plasma concentration of the drugs and extending the duration of their action. The formulation of such a DDS with an extended-release trend not only improves patient compliance, but also minimizes fluctuations in the plasma concentration profile .For decades, different strategies have been used for developing extended-release tablets as a dosage form. For example, matrix-based tablets have been formulated by pharmaceutical companies to create controlled-release forms. However, sometimes, new approaches are needed to enable rapid small-batch production of a tablet with the desired dose and specific kinetic release and meet pharmaceutical economics and quality.3D-printing technology has offered new hopes to achieve this goal because the desired control of patient-tailored tablets and their release kinetics is possible through manipulating the polymer ratio in printer inks . Taking In our study, PVA as the primary polymer and commercially available water-soluble filament were used for HME-based 3D printing . To faciNo drug and polymer interference appeared, as the FTIR spectrum of the 3D-printed tablet corresponded to that of all ingredients. However, the characteristic peaks of the tacrolimus in the FTIR spectrum of the physical drug mixtures with PVA, sorbitol, and magnesium stearate appeared with minor changes. These changes can be attributed to the formation of hydrogen bonds among the hydroxyl groups of tacrolimus, PVA, and sorbitol. Tacrolimus was dissolved in an ethanolic mixture of HPMC to extrude it through the PAM using a second nozzle to prevent drug decomposition under the printing temperature. Obviously, the DSC results indicated that the crystallinity of the tacrolimus remained intact during the printing process. Furthermore, no decomposition occurred under the printing temperature according to the results obtained from the TGA experiment.However, our tablet’s results differed in crystallinity from those of tacrolimus in a disintegrating tablet which was developed by Ponnammal et al. through the hot-melt extrusion of a drug and polymer mixture  at a temperature of about 135 °C, converting to an amorphous form . In contIn the study, we have shown that the 3D-printed tablet can meet the routine physiochemical characterization of traditional tablets in pharmaceutical companies according to USP requirements  as well as intra-process quality control, including weight variation, hardness, and friability tests.According to the dissolution profiles, an extended-release manner for the 3D-printed tacrolimus tablets was found compared to the burst-release trend of commercial tacrolimus capsules. One of the regulatory requirements in developing pharmaceutical dosage forms is establishing the similarity between the dissolution profile of a generic drug and its brand by the similarity factor, f2. Interestingly, a similarity value of more than 60% was statistically obtained between the dissolution profiles of Envarsus XR and 3D-printed tablets, indicating successful formulation of the sustained-release 3D-printed tablet of tacrolimus. Furthermore, our observations showed that the drug release obeys the infill percentage, which is considered for the 3D-printing process. The release profiles were infill%-dependent. The tablet with an infill of 90% had a desired release profile. The 3D-printed tablets were considered stable under accelerated conditions.© and Astagraf XL© tablets, have been developed to simplify the dosing frequency as well as patient compliance for heart, liver, lung and kidney transplantations [Some extended-release dosage forms of tacrolimus, such as Advagrafntations . Furtherntations . The onlntations .Taken together, the extended-release form of tacrolimus tablets developed by an extrusion-based 3D-printing technique in our study conforms to all the obligatory pharmaceutical tests while being economical, versatile, controllable, and relatively effortless to fabricate. The polymers used in our study are also user-friendly, as they can manipulate themselves to load the required amount of drug seamlessly while avoiding thermal degradation throughout the production process and controlling the desired drug release kinetically. The 3D-printed tablet has a high hardness value with no friability, making it suitable for downstream large-scale pharmaceutical processes such as coating, packaging, and handling. Another major advantage of 3D-printing-based manufacturing would be its ability to precisely control dose adjustment, integrate multiple drugs into a pill, and modulate the dissolution rate using different polymer mixtures and infill percentages. In addition, the rapid production technique can provide tailored dosages for point-of-care patients in the personalized medicine field. Specially, the fabrication of the extended-release tablet of tacrolimus using 3D printing in a desired dose would help clinicians to personalize treatment and tacrolimus TDM after transplantation .In the present study, for the first time, we have developed a novel 3D-printed tablet of tacrolimus in a stable extended-release form using HME and PAM-based 3D printing to provide a dosage for preventing graft rejection in the organ transplantation procedure. No drug degradation nor polymer decomposition were observed during the printing process. The kinetic release of the tablet, which is modulated using different polymer mixtures and infill percentages, can enhance patient compliance with one medication per day. The developed tablet met all the acceptance values of USP requirements upon assay, content uniformity, dissolution tests, and microbial tests as well as the features of intra-process quality control. The feasible, economical, and controllable 3D-printing process can bypass the expensive manufacturing steps in pharmaceutical companies and quickly provide a tailored tacrolimus tablet for personalized medicine at the point-of-care.


Anacardium occidentale L.) samples from 5 brands. Multivariate statistical methods, such as principal components analysis (PCA), were employed to analyze the data obtained and establish the provenance of the cashew nuts. While cashew nuts are widely marketed in many countries, no universal method has been utilized to differentiate the origin of these nuts. Our study represents the initial step in identifying the geographical origin of commercial cashew nuts marketed in Vietnam. The analysis showed significant differences in the means of 21 of the 40 analyzed elements among the cashew nut samples from the 5 brands, including 7Li, 11B, 24Mg, 27Al, 44Ca, 48Ti, 51V, 52Cr, 55Mn, 57Fe, 60Ni, 63Cu, 66Zn, 93Nb, 98Mo, 111Cd, 115In, 121Sb, 138Ba, 208Pb, and 209Bi. The PCA analysis indicated that the cashew nut samples can be accurately classified according to their original locations. This research serves as a prerequisite for future studies involving the combination of elemental composition analysis with statistical classification methods for the accurate establishment of cashew nut provenance, which involves the identification of key markers for the original discrimination of cashew nuts.Fingerprinting techniques, which utilize the unique chemical and physical properties of food samples, have emerged as a promising approach for food authentication and traceability. Recent studies have demonstrated significant advancements in food authentication through the use of fingerprinting methods, such as multivariate statistical analysis techniques applied to trace elements and isotope ratios. However, further research is required to optimize these methods and ensure their validity and reliability in real-world applications. In this study, the inductively coupled plasma mass spectrometry (ICP-MS) analytical method was employed to determine the content of 21 elements in 300 cashew nut ( Ensuring food safety, quality, and traceability is critical for public health and consumer confidence . HoweverAnacardium occidentale L.) is a popular nut that has become an important commodity in the global market due to its high nutritional value and versatile uses in the food industry [Cashew nut  solution (65%) and hydrogen peroxide (H2O2) solution (30%) were purchased from Merck, USA. Ultrapure deionized water with a resistivity of 18.2 MΩcm was obtained from the Milli-Q Plus water purification system . Certified reference material (CRM) from CPAChem standard solution 100 mg/L  was used to build the standard curve, and 9 components at 10 mg/L  were used as internal standards.Samples of cashew nuts were collected from 5 different brands, namely, Hanfimex, Hong Loi Thinh, Nha Le, VinaNuts, and Jrai Farm, at large stores and supermarkets in Hanoi. A total of 300 samples (60 from each brand) were collected, labeled with the place of sampling, processing date, and coding number, and stored at room temperature in a fully sealed condition. Nitric acid (HNO3 (65%), 1 mL of H2O2 (30%), and 0.1 mL of internal standard in a Teflon tube and left overnight. The samples were then digested using a preset digestion method for food in MARS 6 . The digested sample was transferred to a 25-mL volumetric flask and made up to the mark with deionized water. Finally, the sample was filtered into a coded falcon tube and was ready for ICP-MS analysis.Each sample was dried and ground to a fine powder, and 0.2 g of the powdered cashew was mixed with 4 mL of HNO7Li, 11B, 24Mg, 27Al, 44Ca, 48Ti, 51V, 52Cr, 55Mn, 57Fe, 60Ni, 63Cu, 66Zn, 93Nb, 98Mo, 111Cd, 115In, 121Sb, 138Ba, 208Pb, and 209Bi) in cashew nut samples. The data indicate that there are variations in the concentrations of these elements across different brands. To elucidate which elements are most significant in distinguishing between brands, statistical analysis methods such as multivariate analysis can be employed.7Li ranged from 0.73 μg/kg DW in Hanfimex to 0.95 μg/kg DW in Jrai Farm. Similarly, the concentration of 27Al ranged from 26.36 μg/kg DW in Nha Le to 68.64 μg/kg DW in Jrai Farm. Some elements, such as 93Nb and 121Sb, were not detected  in some of the samples. However, the LOD for these elements was not specified in the table, which could be considered a limitation of the study. Additionally, the sample size and the statistical significance of the differences between the brands were not reported. The element concentrations from 7Li, 27Al, 44Ca, 57Fe, and 66Zn were the most critical in distinguishing between the brands.As shown in The principal component analysis (PCA) was conducted to identify the variations in the elemental composition of the five brands of cashew nuts. The results of PCA analysis showed that the five brands of cashew nuts can be differentiated based on their elemental composition. The PCA analysis figure clearly indicates that the five brands of cashew nuts are well separated from each other, and each brand is clustered together .R2X cumulative of component 1 (39.64%) and component 2 (20.64%) as shown in The score scatterplot shows separation between brands based on the R2 and Q2 values. The cumulative R2 value is 0.628 and Q2 is 0.5159 for PC1 and PC2 from the PCA model. These results mean that 60.28% and 51.59% of the total variation can be explained and predicted, respectively, based on the first two PCs. The R2 value measures how well the model fits the data, while the Q2 value measures how well the model predicts new data.The summary of the principle component analysis (PCA) in 7Li, 11B, 27Al, 44Ca, 51V, 52Cr, 57Fe, 60Ni, 63Cu, 66Zn, 93Nb, 98Mo, 111Cd, 121Sb, 138Ba, and 208Pb have a significant impact on the PCA model. These variables could be considered as the most important variables in distinguishing between the different brands of cashew nuts based on their elemental composition. The heavy influence of these variables on the PCA model highlights the potential utility of these elements in characterizing and identifying the origin of cashew nuts, which could be important for quality control and food safety purposes. It is worth noting that the loading scatterplot provides valuable information on the variable importance in the PCA model, which can aid in the interpretation of the results and inform future studies  and the AOAC standard to analyze nine elements and five chemical components of cashew nut samples in 2022. The researchers used a combination of elemental profiles and canonical discriminant analysis (CDA) to discriminate the origin of cashew nuts. Potassium (K), magnesium (Mg), and calcium (Ca) were found to be the most abundant elements in cashew nuts, and sodium (Na), calcium (Ca), potassium (K), manganese (Mn), zinc (Zn), and total protein were the best descriptors for cashew nut origin. Additionally, the CDA scatter plot based on a combination of elemental profiles and protein concentration was found to be the best method to visualize the origin of Indonesian cashew nut samples . Other s7Li, 27Al, 44Ca, 57Fe, and 66Zn were the most crucial elements in distinguishing between the brands. The findings also indicated that the elemental composition of cashew nuts could be used to differentiate between the five brands, and the geographic origin could play a significant role in determining the composition. The loading scatterplot identified key variables, such as 7Li, 11B, 27Al, 44Ca, 51V, 52Cr, 57Fe, 60Ni, 63Cu, 66Zn, 93Nb, 98Mo, 111Cd, 121Sb, 138Ba, and 208Pb, that significantly influenced the PCA model and could be considered as the most important variables in differentiating between the brands of cashew nuts based on their elemental composition. The moving charts provided a visual representation of the means and distribution ranges of the variables in the samples, and the Jrai Farm sample had the highest concentration of several elements. Overall, the study suggests that elemental composition analysis can be a useful tool in differentiating between brands of cashew nuts, but further research is necessary to confirm the findings and ensure their statistical significance.In this study, the elemental composition of five different brands of cashew nuts was used to assess the potential influence of geographical origin and brand on their composition. The concentrations of 21 elements were analyzed using multivariate statistical analysis methods including PCA and LDA. The results demonstrated that the concentration of elements varied among the different brands, and some elements were not detected in certain samples. The PCA and LDA revealed that In this study, the content of 21 elements in 300 cashew nut samples from 5 brands was determined using the inductively coupled plasma mass spectrometry (ICP-MS) analytical method. The data obtained were analyzed using multivariate statistical methods, such as principal components analysis (PCA), to establish the provenance of the cashew nuts. Especially, the results showed significant differences in the means of 21 of the 40 analyzed elements among the cashew nut samples from the 5 brands. The PCA analysis indicated that the cashew nut samples can be accurately classified according to their original locations. This study serves as a prerequisite for future studies involving the combination of elemental composition analysis with statistical classification methods for the accurate establishment of cashew nut provenance.


However, the CD3+ cell count, CD4+/CD8+ ratio, neutrophil-to-lymphocyte ratio (NLR), IL-6 and TNF-α levels remained unaffected. Conclusions: The combination of epidural analgesia and general anaesthesia can potentially improve, postoperatively, the NK cell count and CD4+ cell levels in gastric or colon surgery patients. However, the specific impact of TIVA or volatile-based anaesthesia remains uncertain. To gain a better understanding of the immunomodulatory effects of anaesthesia, in this particular group of cancer patients, further well-designed trials are required.Background: Gastric and colorectal carcinomas are associated with increased mortality and an increasing incidence worldwide, while surgical resection remains the primary approach for managing these conditions. Emerging evidence suggests that the immunosuppression induced by the chosen anaesthesia approach, during the perioperative period, can have a significant impact on the immune system and consequently the prognosis of these patients. Aim: This systematic review aims to comprehensively summarize the existing literature on the effects of different anaesthesia techniques on immune system responses, focusing on cellular immunity in patients undergoing the surgical removal of gastric or colorectal carcinomas. There is no meta-analysis investigating anaesthesia’s impact on immune responses in gastric and colorectal cancer surgery. Anaesthesia is a key perioperative factor, yet its significance in this area has not been thoroughly investigated. The clinical question of how the anaesthetic technique choice affects the immune system and prognosis remains unresolved. Methods: Major electronic databases were searched up to February 2023 to May 2023 for relevant randomized controlled trials (RCTs). The study protocol has been registered with Prospero (CRD42023441383). Results: Six RCTs met the selection criteria. Among these, three RCTs investigated the effects of volatile-based anaesthesia versus total intravenous anaesthesia (TIVA), while the other three RCTs compared general anaesthesia alone to the combination of general anaesthesia with epidural anaesthesia. According to our analysis, there were no significant differences between TIVA and volatile-based anaesthesia, in terms of primary and secondary endpoints. The combination of general anaesthesia with epidural analgesia had a positive impact on NK cell counts (SMD 0.61, 95% CI 0.28 to 0.94, I Cancer remains one of the major determinants of morbidity and mortality worldwide . Despite+, CD8+ and CD3+, as well as natural killer cell (NK cell) dysfunction, as well as the excessive proinflammatory responses reflected by an increased lymphocyte-to-neutrophil ratio (LNR), are pivotal characteristics of perioperative cytokine cascade activation [Metastasis is a pivotal event in the progression of cancer, characterized by a complex pathophysiological process , primaritivation . The afoThe aspect that perioperative conditions can influence the growth of carcinomas, as well as the prognosis and tumour progression, has been proposed during the last decades ,8. StudiGastric and colorectal cancers are the most common gastrointestinal tumours, with an increasing incidence worldwide. Considering that a significant number of patients with these tumours undergo multiple surgical procedures, careful consideration should be given to the chosen anaesthesia techniques. Based on the available basic research data, it was hypothesized that perioperative immunosuppression, elicited by the implemented anaesthetic approach, could potentially exert a significant effect on the immune system and consequently the prognosis of patients undergoing surgical resection for malignant tumours. However, the existing data in this area are inconclusive, while there is no meta-analysis examining the effects of anaesthesia on patients with gastric and colorectal carcinoma. The objective of this meta-analysis is to provide clinicians with an up-to-date and comprehensive review of the best available evidence, concerning the impact of anaesthesia techniques, mainly focusing on cellular immunity inflammatory cytokines and the leukocyte-to-neutrophil ratio, in patients with gastric and colorectal cancer. The novelty of the present study lies in the fact that this is the first systematic review and meta-analysis focusing on the effect of various anaesthesia techniques on the immune system of patients undergoing surgery for gastric or colorectal cancer. Anaesthesia stands as one of the most alterable perioperative factors, and thus far, its importance in this field has not been adequately explored. Clinically, the pivotal question of whether the choice of anaesthetic affects the immune system and, consequently, prognosis remains unanswered.The present systematic review and meta-analysis adhered to the guidelines outlined in the PRISMA statement. A systematic and comprehensive search was performed using the electronic databases PubMed Scopus and Cochrane database from February 2023 to May 2023 to identify relevant studies. The study protocol has been registered with PROSPERO under registration number CRD42023441383.This meta-analysis included randomized clinical trials (RCTs) that investigated the impact of diverse anaesthetic techniques on cellular immunity inflammatory cytokines and the LFR in patients undergoing the resection of primary colon or gastric tumours. More specifically, the present study focused on investigating the impact of general anaesthesia using propofol, isoflurane, sevoflurane or desflurane, both as standalone methods or in conjunction with local anaesthesia. Local anaesthesia methods included regional nerve blocks, epidural anaesthesia and spinal anaesthesia. The selected studies were characterized as eligible if they assessed the outcomes of interest, comparing general anaesthesia, alone or in combination, with regional anaesthesia. Studies that did not utilize any of the aforementioned anaesthetic technique combinations were excluded from this meta-analysis. The inclusion criteria for participants were limited to adults only. No limitations were imposed on the utilized analgesia protocols. Moreover, no exclusion criteria were established regarding to the type of surgical procedure , disease stage and comorbidities. Additionally, the duration of hospitalization was not taken into consideration. Language restrictions were not applied, in order to maximize the potential search results and thus minimize potential systematic errors of publication.+ CD4+ CD8+ NK cell count and CD4+/CD8+ ratio, after anaesthesia induction, at the end of the surgical procedure and at 24 h and 72 h post-operation. Moreover, the secondary outcomes included the effect on the IL-6 TNF-a and LFR ratio at the same time points. An available cases approach was implemented, and participants who were excluded from the analysis were assumed to be missing at random.The primary endpoints included the effect of anaesthesia techniques on cellular immunity, as measured by the CD3The relevant records were imported into Endnote 19 and any duplicate entries were eliminated. Two reviewers (GK and TG) independently screened the records, based on their titles and abstracts. Subsequently, eligible studies were assessed in the full text. In cases of disagreements, during the study selection process, a third reviewer (CP) resolved the discrepancies. Data including the year of publication, follow-up period, country of origin, study type, patient characteristics (gender and age), tumour type and stage, anaesthesia type and duration, surgical technique and details regarding primary and secondary endpoints, which were recorded in a predefined format.The risk of bias for the primary outcomes was assessed using The Cochrane Risk of Bias (ROB) tool 2.0. , which eContinuous data were synthesized using Hedge’s estimation method when different scales were used by the individual studies. The results were expressed as standardized mean difference (SMD) and 95% confidence intervals (CIs). For all analyses, final values were utilized, as all studies included in the analysis were randomized controlled trials. When the necessary measures of dispersion were not reported in the studies, the methods described in the Cochrane Textbook were applied  in orderSix studies, including 559 patients in total, met the inclusion criteria. Three RCTs ,20,21, wThree studies ,20,21 exThree RCTs ,23,24 inThe systematic risk of bias for all six studies was evaluated by two independent reviewers  using ROB 2.0. The results are presented in a.NK cell counts: propofol versus sevoflurane2 16.09%) (2 0.0%)  , as well(2 0.0%) , no statb.NK cell counts: general anaesthesia versus general anaesthesia plus epidural anaesthesia2 0.0% and SMD 0.49, CI 95% 0.16 to 0.82, I2 0.0%)    . On the 2 58.1%) . In bothd.+, CD4+, CD8+ and CD4+/CD8+: general anaesthesia versus general anaesthesia plus epidural anaesthesiaCD3+ cell count 72 h after the surgery     , while C 63.11%) . In relau et al.  reported2 72.4%) .a.NLR2 92.71% and SMD −0.12, CI 95% −0.41 to 0.17, I2 36.12%)  (2 98.2%) (2 98.79%)  . In a pr2 98.2%) . Two RCT 98.79%) .c.TNF-a2 95.9%) (2 92.6%) (2 90.8%)  . In a po2 92.6%) . The ana2 90.8%) .+, while CD3+, the CD4+/CD8+ ratio, NLR IL-6 and TNF-α remained unaffected.The purpose of this systematic review and meta-analysis was to investigate the impact of different anaesthetic techniques on immune system changes, considering cellular immunity inflammatory cytokines and the leukocyte-to-neutrophil ratio. From the limited number of relevant RCTs incorporated in our analysis, no particular differentiation occurred among TIVA and volatile-based anaesthesia, regarding the primary and secondary endpoints. The combination of general anaesthesia with epidural analgesia exerted a favourable effect on the NK cell count and CD4T lymphocytes and NK cells have potential antitumor effects, controlled by several mechanisms in surgical patients. NK cells are related to the antibody-dependent cytotoxicity that mediates both the innate and adaptive immunity ,25. MoreIn the present study, the analysis between TIVA or volatile effects suggested a potential beneficial effect of propofol, after the induction of anaesthesia, as well as 24 h postoperatively, which, however, could not be statistically confirmed. The results should be carefully evaluated since they could not be solidified though sensitivity analysis and due to the fact that great heterogeneity was noted. In addition, the analysis of studies characterized by a low risk of bias agreed with the main results. Notably, our meta-analysis revealed that the combination of general and epidural anaesthesia can significantly increase the NK cell count, an effect being valid up to 72 h postoperatively. Regarding the beneficial effect of propofol, compared with volatile agents, our results agree with an older systematic review and meta-analysis, involving patients with cancer , which p+ count was attenuated 24 h postoperatively in the study of Chen Yi Jiao et al. [+ count augmentation in the immediate postoperative period and 24 h later. Our pooled analysis revealed that propofol slightly reduced the CD8+ count during the immediate postoperative period, in comparison to sevoflurane, and that this reduction was reversed after 24 h, when a slight increase was apparent. However, statistical significance was not achieved.Moreover, the results from two studies on T lymphocyte activity were controversial considering propofol or sevoflurane administration. The beneficial effect of propofol on the CD8o et al. , while io et al. , sevoflu+ count. However, the combination of anaesthetic techniques did not increase the CD4+/CD8+ ratio significantly, even 72 h after the surgery. According to the literature, CD3+, CD4+ and CD8+ are categorized as tumour-infiltrating. Tumour-infiltrating lymphocytes (TILs) are the major type of infiltrating immune cells [The pooled results of two studies, comparing general anaesthesia alone or in combination with epidural anaesthesia, revealed a beneficial clinical effect of the combined technique, inducing an elevated or stable CD4ne cells ,36. The ne cells . It is hne cells . The comne cells ,40.With regard to the secondary outcomes of our analysis, no profound impact of the anaesthesia regimen or anaesthetic technique on NLR could be demonstrated. Recent studies have proven that NLR, which is a systemic inflammatory response biomarker, can be used as a predictor of survival in colorectal or gastric cancer patients . AlthougMoreover, recently, great progress has been made in understanding the role of circulating cytokines in cancer prognosis. Published data revealed that high IL-6 levels can be an independent biomarker for a bad prognosis in patients with gastrointestinal cancer, while the results for colorectal cancer were controversial . Our metAmong cytokines related to immunomodulation, TNF-a has been recognized as the most significant proinflammatory cytokine involved in the pathway of carcinogenesis . Recent + cells, decrease in the CD4+/CD8+ T cell ratio and decrease in the absolute number of NK cells [Another aspect to consider is the role of perioperative transfusions, which are routinely conducted during surgical procedures. Allogeneic blood transfusions have been demonstrated to impact various immune functions in recipients, including a reduction in the total number of lymphocytes, decrease in the number of CD4NK cells . TransfuMore specifically, TRIM could be induced through the secretion of Type Th2 immunosuppressive cytokines, as well as the release of Type Th1 pro-inflammatory cytokines released by WBCs during the storage of blood products ,49. The Additionally, it is important to consider the variation in analgesia protocols across the studies during surgical procedures. The diverse forms of analgesia exert a significant influence on perioperative immunomodulation. Notably, opioids consist of the most frequently administered analgesics, extensively utilized in the context of cancer surgeries. It has been established that opioids can suppress both cellular and humoral immunity, thereby promoting a state of immune suppression ,53. Thei+ cytotoxic T cells and NK cells, all of which contribute to facilitating tumour immune escape. Consequently, medications that target the production of PGE2 and COX-2 are linked to potential immunomodulatory effects [Non-steroidal anti-inflammatory drugs (NSAIDS) are another category of analgesics that should be taken into consideration. Enzymes that regulate the production of prostaglandins and cyclooxygenase are often overexpressed in malignancies . The COX effects . A recen effects . DespiteWe acknowledge that the present study has certain limitations, which should be taken into consideration. The analysis was based on a limited number of studies, and significant heterogeneity was observed among them. In addition, the included studies varied also in terms of methodology, population types of surgery disease stage and the anaesthetic techniques used. To address the above difficulties, an available-case analysis was conducted, while the last observation was carried forward for continuous data. Albeit these statistical approaches are well-documented and yield reliable results, caution should be exercised when interpreting the findings. Moreover, it is worth mentioning that the main results of this study may be susceptible to statistical bias as the scarcity of data prevented us from conducting the predetermined sensitive analysis.In the future, the results of our analysis can be combined with the impact of anaesthetic agents and practices during operations for other malignancies, in order to finally reveal if there is an ideal technique for cancer surgical patients. However, the only way to safely conclude on certain anaesthetic plans used during the surgical excision of a tumour is through an analysis of existent high-quality randomized controlled trials.+ levels in a subset of patients subjected to gastric or colon surgery, while TNF-α and IL-6 levels, as well as CD3+, the NLR and the CD4+/CD8+ ratio remained unaffected. Of note, the clear-cut role of TIVA or volatile-based anaesthesia in this setting could not be ascertained. However, due to the heterogeneity and inconsistent results of the limited available evidence on this topic, no definite conclusions could be drawn. Thus, our study serves as a call for future well-designed comparative trials with homogeneous methodologies to elucidate the potential immunomodulatory role of the applied anaesthetic practice in these subsets of cancer patients.Given the unique role of immunomodulation in cancer progression and the distinctive need to elucidate possible modifiers of this process, the supplementation of general anaesthesia with epidural analgesia might exert a favourable effect on the NK cell count and CD4


N = 2,297). It found that use of DHTs had a significantly positive effect on Chinese youths' healthy lifestyles and mental health, with behavioral regulation as a mediator. However, social interactions of DHTs were negatively associated with their mental health. The findings contribute to a better guidance on health promotion, as well as the enhanced design of DHTs' products.The rapid development of intelligent technologies coupled with the stay-at-home trends in the Post-COVID-19 Era has significantly changed youth's health behavior as well as reshaped their lifestyles. Digital health technologies (DHTs) have been more and more used for health management among youngsters. However, little was known about the use of DHTs among youths and its consequences on their health, especially in developing countries like China. Inspired by behavior intervention technology (BIT) model, this study examined the underlying mechanisms of use and social interactions of DHTs on Chinese adolescents' and youngsters' healthy lifestyles and mental health, using a representatively national survey among high school and freshman students in China ( At the end of 2019, a sudden epidemic swept the world. During the COVID-19 pandemic, the imposition of lockdown measures was unprecedented and dramatically changed the common life of the public . SeveralMeanwhile, there has been increasingly academic interests in digital health technologies  as tools for health management in recent years . EspeciaAlthough there were several studies which have explored the relationship between the use of DHTs and users' health behaviors or healthy lifestyles , 15, theIn addition, most of the prior findings aimed at adults and used convenience samples of adults , while tTherefore, the aim of this study was to explore the impact and underlying mechanisms of the use and social interactions of DHTs on adolescents' and youngsters' healthy lifestyles and mental health, based on a national sample consisting of Chinese students.In the age of digital technologies, more and more adolescents are using digital technologies to explore health topics and conduct health management . NationaThousands of “healthcare and fitness” apps for mobile clients provided technical support for health management . Fitbit DHTs usually consisted of mHealth, wearables, and digital devices for health management and promotion –36. AmonA healthy lifestyle is effective in reducing the risk of serious illness or premature death . It has Prior studies showed that the use of digital interventions may have the potential to improve individuals' diet, physical activities, sleep, and weight control , 20, 30.Furthermore, previous studies have shown that DHTs combined with social influence were effective in increasing users' levels of physical activities , 48. ShaTherefore, a set of following hypotheses were articulated as  shows:H1a: Use of DHTs is positively correlated with healthy lifestyles.H2a: Social interactions of DHTs are positively correlated with healthy lifestyles.H3a: Use of DHTs is positively correlated with mental health.H4a: Social interactions of DHTs are positively correlated with mental health.With the rapid development of science and technology, technological innovations continued to reshape people's cognition and life. The behavior intervention technology (BIT) model explained changes in healthy behaviors caused by technical interventions. BIT referred to behavioral and psychological interventions through devices  and software  , aiming It revealed that one's self-regulation played an important role in the process of technological products triggering changes in healthy behaviors. Self-awareness, self-management, and self-efficacy were also important self-binding strategies of BIT for successful interventions to promote healthy behavioral changes , 54. StuBehavioral regulation included introjected regulation and identified regulation. Introjected regulation implied that the behavior was done for controlling reasons (to avoid guilt or to please others), whereas identified regulation implied that one has accepted the behavior as important and meaningful, i.e., self-determined . Both ofThese four hypotheses were put forward accordingly:H1b: Behavioral regulation mediates the relationship between use of DHTs and healthy lifestyles.H2b: Behavioral regulation mediates the relationship between social interactions of DHTs and healthy lifestyles.H3b: Behavioral regulation mediates the relationship between use of DHTs and mental health.H4b: Behavioral regulation mediates the relationship between social interactions of DHTs and mental health.As mentioned above, inspired by BIT model, we examined the underlying mechanisms of use of DHTs on adolescents' healthy lifestyles and mental health. To fill the gaps in existing studies, we incorporated the use and social interactions of DHTs as predictors and behavioral regulation as a mediator in this work. M = 18.71, SD = 1.814). Sixty-six trained investigators participated in the questionnaire survey. Finally, we received 3,330 responses from 31 provinces and metropolitans across the whole China. After removing respondents that have not used any DHTs or missed more than 10% of the items, a total of 2,297 responses were obtained, with effective rate of 68.98%.To test the hypotheses, a nationwide questionnaire survey was conducted in China from May 2021 to June 2021, using a cluster-randomized sampling method to obtain representative national samples. The subjects of the survey were adolescents and youngsters aged from 15 to 24 years old  from high schools and colleges were invited for a pretest. After they completed the questionnaire, an interview was conducted to collect their opinions and suggestions. Based on their feedback, some problematic items were modified accordingly to improve the accuracy of the questionnaire, and then the scales were finalized for this study. One high school and one university were selected as the target schools in each of the 31 provinces or metropolitans across China, via a cluster-randomized sampling method. Students at the target schools were interviewed anonymously in their own classrooms. All the participants completed the survey after the trained interviewers provided detailed instructions to them and all the informed consents were obtained from the schools, teachers, and students. Investigational sessions lasted approximately 20 min, in which cross-sectional data were collected.The gender, age, and grade of the students were included in this survey. Besides, all the participants were asked to calculate and report their own BMI, which was measured by their height and weight.To measure the extent of participants' the use of DHTs, they were asked two questions: “For the above-mentioned devices/APPs that you used most frequently, approximately how many times did you use them per week in the past month?” and “How many minutes did you use each time?” Participants filled in the real numbers based on their usage, and the data were analyzed by dividing the answers into six levels (1 to 6) in equal intervals. The final score of DHTs use was calculated based on the mean of two items.Participants' social interactions of DHTs were measured by asking: “In the past month, how often did you share your sports-related situations  via the devices/apps on social media platforms ?”, and “In the past month, how often did you talk to others about your use of the devices/apps and other related topics?”, with the answers ranging from 1 = never, 2 = once, 3 = 2–3 times, 4 = at least once per week, to 5 = two or more times per week. The two items were averaged to form the score of social interactions of DHTs. A higher score represented higher level of social interactions.M = 2.70, SD = 0.76, α = 0.88).Behavioral regulation was measured by 12 items derived from the Behavioral Regulation in Exercise Questionnaire (BREQ) , which iM = 3.02, SD = 0.66, α = 0.88).This construct was measured by 23 items, which were modified based on the health Promotion Lifestyle Profile Scale (HPLP-S) , consistM = 10.21, SD = 5.60, α = 0.81). Higher scores represented more severe depressive symptoms.Mental health was measured by the 10-item Chinese version of Center for Epidemiologic Studies Depression Scale (CESD-10) , 60, whiDescriptive statistics were used to assess respondents' demographic characteristics  and the overall usage of DHTs. Pearson correlations (two tails) were used to examine the correlations between independent variables and dependent variables. In order to test the mediation effects of behavioral regulation, four mediation models were employed. Descriptive statistics and Pearson correlations were calculated with IBM SPSS 25; mediation effect analyses were tested using the PROCESS version 3.5 (Model 4) proposed by Hayes ; standarSD = 1.814). The majority of respondents' BMI was in the healthy category (62.1%). As regards to the use of DHTs, “KEEP” had the highest usage rate (75.9%), followed by smartwatches (39.1%), Sports World Campus (13.3%), etc. In addition, exercise record  (62.8%) and exercise guidance (46.6%) were the most frequently used functions.t = 9.333, p<0.001) and social interactions of DHTs  positively predict healthy lifestyles, which confirmed Hypothesis 1a and 2a. Analyzing the indirect effects, results revealed that behavioral regulation mediated the relationship between use of DHTs and healthy lifestyles , also the relationship between social interactions of DHTs and healthy lifestyles . Hence, Hypotheses 1b and 2b were supported. Nevertheless, the results also suggested that even after accounting for the mediating role of behavioral regulation, both use of DHTs  and social interactions of DHTs  still had a positive impact on healthy lifestyles. Behavioral regulation accounted for 42.69% of the total effect in model 1 and 72.01% in model 2. The detailed results were indicated in As for model 1 and model 2, the results showed that both use of DHTs , thus failing to support Hypothesis 3a. But social interactions of DHTs had a positive impact on depression , which meant negative association with mental health. The result was contrary to hypothesis 4a. Analyzing the indirect effects, results revealed that behavioral regulation mediated the relationship between use of DHTs and mental health , also the relationship between social interactions of DHTs and mental health . Hence, Hypotheses 3b and 4b were supported. Nevertheless, the results also suggested that after accounting for the mediating role of behavioral regulation, use of DHTs had a negative impact on depression , which meant positive association with mental health. Meanwhile, social interactions of DHTs still had a positive impact on depression , which meant negative association with mental health. Behavioral regulation accounted for 22.75% of the total effect in model 3 and 46.24% in model 4. The detailed results were shown in Regarding model 3 and model 4, the result showed that use of DHTs couldn't directly predict mental health . Written informed consent to participate in this study was provided by the participants' legal guardian or themselves. Participation was completely voluntary, and the participants could choose to quit at any time for any reason during the process of answering the questionnaire. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XL contributed to conceptualization, methodology, investigation, resources, writing—original draft preparation, writing—review and editing, project administration, and funding acquisition. MZ contributed to formal analysis, investigation, data analysis, and writing—original draft preparation. All authors have made a substantial, direct, and intellectual contribution to the work, and agreed to the published version of the manuscript.


Delonix regia trypsin inhibitor (DrTI). This protein inhibits serine proteases (trypsin) and enzymes directly involved in coagulation, such as plasma kallikrein, factor XIIa, and factor XIa. In this study, we evaluated the effects of two new synthetic peptides derived from the primary sequence of DrTI in coagulation and thrombosis models to understand the mechanisms involved in the pathophysiology of thrombus formation as well as in the development of new antithrombotic therapies. Both peptides acted on in vitro hemostasis-related parameters, showing promising results, prolonging the Partially Activated Thromboplastin Time (aPTT) and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid. In murine models, for arterial thrombosis induced by photochemical injury, and platelet-endothelial interactions monitored by intravital microscopy, both peptides at doses of 0.5 mg/kg significantly extended the time of artery occlusion and modified the platelet adhesion and aggregation pattern with no changes in bleeding time, demonstrating the high biotechnological potential of both molecules.Several plant protein inhibitors with anticoagulant properties have been studied and characterized, including the  According to the World Health Organization, atherothrombotic diseases—atherosclerosis, thrombosis, and heart attack, among others—represent one of the greatest health challenges, accounting for more than 25% of deaths worldwide, with 80% of deaths occurring in underdeveloped and developing countries . Atherot®), glycoprotein IIb/IIIa inhibitors (Abciximab®), protease-activated receptor antagonists, and ADP receptor antagonists [Antithrombotic therapies are classified as either anticoagulants or antiplatelet agents depending on their target. Even before the introduction of the chemical principles of clinical therapeutics and random trials, anticoagulants, such as heparin and warfarin, and antiplatelet drugs, such as acetylsalicylic acid (ASA) and dicoumarol, were already utilized as antithrombotic therapies, even though the mechanism of action has not been elucidated . Nowadayagonists . The bigagonists .Delonix regia trypsin inhibitor (DrTI) is a Kunitz-type inhibitor with a molecular mass of approximately 20 kDa and two loops formed by four cysteine residues. Unlike other Kunitz inhibitors, which contain either arginine or lysine, glutamic acid is found at the reactive site position  [app = 2.2 × 10−8 M), human plasma kallikrein , factor XIIa , and factor XIa , as well as ex vivo ADP-mediated platelet aggregation [Protease inhibitors from plants have been widely studied and differ in potency and specificity. Kunitz-type inhibitors have been found in several plants and have been characterized for decades for their ability to act as antimicrobial, antitumor, and anticoagulant agents ,9,10,11.hibitor) . The strhibitor) . RegardiIn this study, we describe two novel peptides whose design is based on the structure of the protease inhibitor, DrTI. These peptides inhibit coagulation proteases, affect platelet activity both in vitro and in vivo, and prolong the time for arterial occlusion in murine models of thrombosis, doing all of that without impairing hemostatic parameters, such as bleeding time and ex vivo coagulation tests, and showing great biotechnological potential.Bovine trypsin was purchased from Roche  and human plasma kallikrein (PKa) was purified in the Laboratory of Chemistry and Protein Function using the protocol established by Prof. Dr. Maria Luiza Vilela Oliva and colleagues in 1982 .®). Rose Bengal  and rhodamine 6G were purchased from Sigma Aldrich . ADP, arachidonic acid, and collagen I were obtained from Chrono-log .The substrates Boc-Glu(Obzl)-Ala-Arg-AMC.HCl, Bz-DL-Arg-pNA.HCl and H-D-Pro-Phe-Arg-pNa were purchased from Bachem . Thromborel S, thrombin, and Dade actin-activated cephaloplastin were obtained from Dade Behring , and heparin was obtained from Roche , and synthesized by Watsonbio , were used and their homogeneity, purity, and concentration were evaluated using reversed-phase high-performance liquid chromatography. Both peptides are mirror images, and the pDrTI-RI peptide sequence is the inverse of the pDrTI sequence . The animals were maintained at 23 °C on a 12 h light/dark cycle with water and food ad libitum. The project was approved by the Ethics Committee on the Use of Animals of UNIFESP (CEUA UNIFESP) under number 7922240819. The studies were carried out following international principles of animal use, such as those established in the Manual on Care and Use of Laboratory Animals  and Federal Law 6638 of 8 May 1979. The animals were treated in accordance with the ethical principles of animal experimentation developed by the National Council for the Control of Animal Experiments (CONCEA) and established by the Canadian Council on Animal Care and the Guide for the Care in Use of Animals from the NIH. After experimentation, the animals were euthanized and disposed of, as determined by the Committee on Ethics in the Use of Animals (CEUA).g for 10 min at 25 °C to obtain platelet-rich plasma (PRP), which was then centrifuged again at 5000× g for 10 min at 25 °C to obtain plasma poor in platelets (PPP). Plasma was aliquoted and stored at −80 °C for further testing. Platelet bags, kindly provided by the Beneficent Association of Blood Collection , were used for the platelet aggregation assays. Basal platelet activity was evaluated before all experiments.Blood from healthy volunteers was collected by venipuncture of the basilic vein in a syringe containing 1/10 volume of 3.8% sodium citrate solution. The collection was performed slowly to avoid blood hemolysis. Blood was centrifuged at 3000× The inhibitory activity of the peptides and the purified protein was measured using clotting enzymes and their chromogenic substrates in 96-well plates at 37 °C. Briefly, enzymes were incubated with peptides or saline for 10 min. The reactions were initiated by the addition of the substrate, and the reaction was continuously monitored using a plate spectrophotometer.2. Inhibitors were incubated for 10 min with the enzyme at a concentration of 25 µM. The substrate was then added, and the reaction was monitored for 30 min with readings every 5 min. Substrate hydrolysis was verified at 405 nm using a microplate reader (Spectra CountTM). For confirmation, inhibition was performed using endpoint readings with increasing concentrations of the inhibitors under study.For trypsin inhibition (37 nM), the substrate Bz-DL-Arg-pNA.HCl (1 mM) was used in 0.05 M Tris-HCl buffer (pH 8.0) containing 0.2% CaClFor PKa inhibition (14.7 nM), the substrate HD-Pro Phe-Arg-pNa (2.5 mM) was used in 0.05 M Tris-HCl buffer (pH 7.4) containing 12 mM NaCl and 1 mg/mL bovine serum albumin. The enzyme was incubated with the inhibitors at a concentration of 25 µM for 10 min and then the substrate was added. Reactions were performed for 30 min, with readings every 5 min. Absorbance was read at 405 nm using a microplate reader (Spectra CountTM). Inhibitors were also evaluated at increasing concentrations in assays with endpoint readings.For the in vitro coagulation assays, the pool of blood plasma was used as described previously, measurements were performed in triplicate, and the results were expressed relative to the control. Assays were performed using a semi-automatic coagulometer . For the ex vivo assays, the compounds were administered intraperitoneally. After 10 min, the animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). Once anesthesia was confirmed, blood was collected by cardiac puncture with a 1 mL syringe containing 3.8% sodium citrate in a 1:9 ratio, and the animals were euthanized. The PPP was obtained as described in 2 was added at 37 °C. The reaction was timed and the results were expressed in seconds, with a maximum time of 300 s.Activated partial thromboplastin time (aPTT) was determined using a semiautomatic coagulometer  using activated cephalin from rabbit brain containing celite as an activator. PPP (50 µL) was pre-incubated at 37 °C for 120 s with 50 µL of cephalin and 50 µL of different concentrations of the inhibitors. After pre-incubation, 50 µL 0.025 M CaCl2 1.00 mM, MgCl2 1.00 mM, NaH2PO4 12 mM, HEPES 5 mM, and Glucose 5.56 mM, pH 7.4), and its concentration was adjusted to approximately 3.5 × 105 platelets/mL. Different concentrations of the inhibitors were incubated with PRP in 500 µL cuvettes under constant agitation at 37 °C for 10 min. At the end of the incubation period, an agonist  was added, and the reaction was monitored by measuring the transmittance at 595 nm.For platelet aggregation, experiments were performed using a Chrono-log 700 aggregometer. Once the activity was confirmed, the platelets were diluted in HEPES-Tyrode buffer . The injured artery was then monitored for 12 min (defined as the maximum fluorescence time) using an intravital fluorescence microscope (Carl Zeiss Imager.A2) coupled with an Axiocam HSm camera . After obtaining images, the animals were euthanized.After anesthesia, the carotid arteries of the animals were isolated and dissected as described above. After dissection, the substances under study were administered intravenously through the retro-orbital plexus, and 5 min later, rhodamine 6G  (1 mg/kg) was also administered via retro-orbital injection in the opposite optic cavity to label mitochondria from platelets and leukocytes. For the aspirin control, aspirin diluted in acidic medium was administered by gavage 30 min before the dissection procedure. The arterial injury was induced after 5 min by applying, for 2 min, a piece of filter paper (2 × 1 mm) saturated with 15% FeClThe procedure described by Perzborn et al. , with sop < 0.05). Data are reported as mean ± standard error. Statistical summary data were graphically presented using box plots. All analyses were performed using SPSS version 20 .As the original data were not normally distributed, all analyses were based on nonparametric methods. Statistically significant differences were determined using Kruskal-Wallis tests complemented by Dunn’s multiple comparisons with significance set at 5% , and heparin left the plasma incoagulable A. The reThe inhibitory activity of the pDrTI-RI peptide on intrinsic coagulation pathway enzymes and possibly FXIa was determined in the plasma inhibition assay. When ex vivo aPTT tests were performed, none of the peptides were able to change the clot formation time C at the As already described in the literature, DrTI can inhibit ADP-mediated platelet aggregation ex vivo in blood aggregometry . The inhp < 0.05 in the t-test with Welch’s correlation. Heparin, usually used in clinics for anticoagulation, showed no significant difference compared with the negative control  and platelet behavior profile for DrTI and aspirin were defined . We obset manner . Anothert manner .Both peptides demonstrated the same pattern of platelet behavior as that observed for the protein . AlthougPeptides have already been shown to prolong the occlusion time in arterial thrombosis, do not change ex vivo coagulation assays, and appear to act mainly on platelets and thrombus propagation, a combination of factors favorable to the absence of bleeding. With this in mind, bleeding time assays, or induced bleeding time, was performed in mice using these peptides .While heparin made the mice uncoagulable, neither peptide significantly altered the time required to stop the bleeding. The pDrTI-RI peptide appeared to increase the coagulability of the animal in this model, which should be further investigated, while the pDrTI peptide maintained, on average, the same time required for the untreated animal’s blood to clot.Kunitz-type inhibitors, which have been characterized for decades, are present in several plants, have been widely studied, differ mostly in potency and specificity of inhibition, and can act as antimicrobial, antitumor, and anticoagulant agents in vivo ,8,10,11.Among these inhibitors, DrTI stands out for its ability to inhibit PKa and FXIa, which are involved in several biological pathways, such as the intrinsic coagulation pathway, complement system, renin-angiotensin system, and kallikrein-kinins system, as well as in fibrinolysis and inflammation . In receDrTI has notable characteristics. In addition to inhibiting proteins that are at the center of attention in antithrombotic research, DrTI is capable of inhibiting platelet activity and acting as an arterial antithrombotic without altering bleeding time, which is considered the greatest limiting factor of these therapies. However, owing to the size of proteins, the importance of the three-dimensional protein structure to function, which is maintained by weak non-covalent forces, and the cost of extraction and purification in large quantities makes the use of proteins for basic characterization and clinical use an expensive process. Other challenges faced in the search for proteins as biopharmaceuticals are their multifaceted mechanisms of action as well as the potentially high number of degradation products, which can act in several parallel biochemical processes .Consequently, the Laboratory of Chemistry and Protein Function group at UNIFESP, led by Prof. Dr. Maria Luiza Vilela Oliva, explored protein fragments capable of maintaining antithrombotic action without increasing bleeding, avoiding most of the limitations found in the use of proteins, which is the case for the peptides studied in this work.In recent years, owing to their size and the balance between structural rigidity and versatility, peptides have become promising candidates as inhibitors with satisfactory affinity and specificity and fewer side effects and toxicity. Deciphering and characterizing the mechanisms of interaction between peptides and proteins is imperative for the development of strategies to interfere with the action of endogenous proteins or to increase the affinity and specificity of existing strategies. Several methods have been used to design peptides using biological approaches, such as phage display, or in silico approaches, such as molecular docking .Usually, for the design of peptides with the same activity as the natural inhibitor, the structure of the binding site of the protein is used as a basis; however, for the peptides used in this study, after analyzing the primary structure of DrTI, we found a tetra-peptide sequence—Ser–Pro-Phe-Arg—similar to that found in the vasoactive peptide bradykinin . This leBoth peptides are mirror images, and the pDrTI-RI peptide sequence was the inverse of the pDrTI sequence. However, pDrTI-RI was synthesized with D-amino acids, making pDrTI-RI and pDrTI enantiomers. The use of d-amino acids is a peptide modification strategy aimed at greater stability, since these amino acids rarely serve as substrates for endogenous proteins, making them more resistant to proteolysis and increasing their half-life .In this way, the first phase of the work to characterize the action of these peptides consisted of protease inhibition assays, in which the native protein has already been characterized by its inhibitory activity: bovine trypsin, PKa, and FXIa. Our results demonstrated that the two peptides were unable to inhibit these three enzymes at concentrations similar to those used by the protein; however, at high concentrations, the peptide pDrTI inhibited PKa activity and pDrTI-RI inhibited FXIa activity. Some PKa inhibitors are also capable of inhibiting FXIa activity, as the proteins are 58% similar . HoweverThe aPTT coagulation assay is widely used clinically and in basic research to assess coagulation changes. aPTT is a screening test sensitive to changes in the intrinsic coagulation pathway and can indirectly measure the activities of factors II, V, VIII, IX, XI, XII, kallikrein, high molecular weight kininogen, and reduced fibrinogen concentration . The actIn the case of platelet aggregation, the two peptides were able to inhibit the aggregation induced by ADP and arachidonic acid at concentrations similar to those of the native protein, with greater effectiveness compared to the native protein. The action of these peptides on platelet aggregation may occur in a pathway common to these two agonists after platelet activation, such as the release of α-granules or thrombin generation, or acting separately on the two pathways, which may be investigated by measuring the levels of thromboxane B2 generated .In view of the protease inhibition and platelet aggregation results, the concentration for thrombosis models was set at 100 µM, equivalent to a dosage of 0.5 mg/kg. According to the data obtained in this work, at this concentration, the peptides are capable of inhibiting platelet aggregation but do not act on coagulation proteases. The pDrTI peptide took 88.6 ± 9 min for complete occlusion and the pDrTI-RI peptide took 97.8 ± 22 min, much higher than the control group, whose time to total artery occlusion was 53.0 ± 7 min, while heparin, used as a positive control, was 68.5 ± 14 min. On average, the peptides increased the time required for artery occlusion by two-fold, comparable to other protein inhibitors previously described.Arterial thrombi are mostly formed by platelets, and a decrease in platelet aggregation is a good alternative to alleviate the pathological process, as has already been demonstrated by the use of drugs such as aspirin and clopidogrel . In an aThe biggest challenge and limiting factor in antithrombotic therapies is bleeding. Currently, most, if not all, antithrombotic drugs used in the clinic have bleeding as one of their side effects. The search for compounds capable of protecting against thrombosis without increasing the risk of bleeding is a holy grail of basic research on this subject . The datIn conclusion, the DrTI-derived mimetic peptides studied in this work maintained the main characteristics of the protein, its action on platelets, and the increased time required for arterial occlusion by thrombus formation with no prolongation of bleeding time. Although the mechanism of action of these peptides has not been characterized, their potential as possible antiplatelet drugs or as a biotechnological tools for the study of platelets is remarkable.


Common mental disorders are highly prevalent in the population, affecting people across all regions of the world. In Ethiopia, mental disorders are the leading non-communicable disorders. World Health Organization (WHO) report shows that 4,480,113 (4.7%) and 3,139,003 (3.3%) people in Ethiopia are estimated to suffer from depression and anxiety, respectively. However, there are only limited studies conducted on common mental disorders in Africa including Ethiopia. Even if there was a previous study conducted among Harari regional state residents 7 years back, nowadays there are many possible factors that could increase the prevalence of common mental disorders in the community like the novel coronavirus pandemic, ethnic war, and current socio-economic crises in Ethiopia.To assess the prevalence of common mental disorders and associated factors among adult residents of Harari regional state, eastern Ethiopia.p < 0.05 was used to identify factors associated with common mental disorders.A community-based cross-sectional study was conducted from March 1 to March 30, 2022. A systematic random sampling technique was used to select 1,192 study participants. Data were collected by interviewer-administered structured and semi-structured questionnaires. A common mental disorder was assessed by the Self-Reporting Questionnaire of 20-Item (SRQ-20) questionnaire. Data were entered into Epi Data version 3.2 and exported to Stata Version 16 for analysis. The multivariable binary logistic regression analysis with a 95% confidence interval and From a total of 1,192 eligible participants, 1,168 responded to this survey, giving a response rate of 97.98%. The prevalence of common mental disorders among adults in the Harari Region of this study was 21.31% . Being female with Adjusted Odds Ratios , single , divorced , widowed , unemployed , having family history of mental illness  and age greater or equal to 55 years  were statistically associated with common mental disorders.The prevalence of common mental disorder in the study area was sharply increased. Being female, single, divorced, widowed, family history of mental illness, being unemployed and age greater or equal to 55 years old were statistically positively associated with common mental disorder. Considering these determinants, screening, early identification, and providing appropriate intervention for common mental disorders in the community should be of great concern. Mental health is a state of well-being in which every individual realizes his or her potential, can cope with the normal stresses of life, can work productively and fruitfully, and can contribute to her or his community . Common Common mental disorders are highly prevalent in the population, affecting people across all regions of the world . The gloThere are limited studies conducted on common mental disorders in Africa. Besides, the available studies reported different figures from different countries. A population-based epidemiological survey of a rural area in Kenya revealed the point prevalence of CMD was 10.8%, largely comprising mixed anxiety-depression 6.1%), panic disorder (2.6%), generalized anxiety disorder (1.6%), and depressive episodes 0.7%) , and chewing Khat , 11, 18.Generally, even if there was a previous study conducted among Harari regional state residents 6 years back , nowadayA community-based quantitative cross-sectional study was conducted from March 1 to March 30, 2022, in Harari regional state, which is located 510 km away from the capital, Addis Ababa. The region is divided into nine districts, with three of them being rural and six being urban. The urban districts are subdivided into 19 kebeles (the lowest administrative division in the country), and the rural districts are subdivided into 17 peasant associations .Harari regional state residents were a source population. All residents living in randomly selected kebeles of Harari regional state with age greater or equal to 18 years were a study population. Residents living in randomly selected households as heads or any other household members greater than or equal to 18 years of age and residents living more than 6 months and available during data collection were included in the study. Peoples who left the house for some reason and who were seriously ill during the data collection period were excluded.n = the minimum sample size required, p = the estimated proportion of CMD, z = the standard value of confidence level of alpha = 95%, d = the margin of error between the sample and the population (0.03). For this study p = 14.9%  was used  method. Then, from selected kebeles, 1,192 households were allocated proportionally. Each study unit (HH) was selected using a systematic random sampling method. Then, the eligible individual within the selected household was selected using a simple random sampling method.Face-to-face interviews were used to collect data using a semi-structured questionnaire. The questionnaire contains four parts, which are socio-economic characteristics of the patients adapted and modified from reviewing similar literature, and clinical, psychosocial, and substance-related factors. Common mental disorders were assessed by using the Self-Reporting Questionnaire of 20-Item (SRQ-20) developed by the World Health Organization. It has 20 questions that are answered by YES or NO with codes “1” which represent the presence of a symptom, and “0” if the symptom is absent. If the submission of the respondent answers more than six of the twenty questions the respondent was considered to have common mental disorders –21. WithSocial support was assessed by the Oslo social support scale (Oslo-3) which contains three items. It is a 3 item questionnaire, commonly used to assess social support and it has been used in several studies. The sum score scale ranged from 3 to 14, which had three categories: poor support 3–8, moderate support 9–11, and strong support 12–14 , and it Substance-related factors were assessed by Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST), which is a brief screening questionnaire developed and validated by the World Health Organization (WHO) to find out about people’s use of psychoactive substances. It was used to assess the current and ever substance use history of the participants . Data weStatus of common mental disorders (yes/no).Socio-demographic variables , clinical factors , substance-related factors , and psychosocial factor .Data collectors and supervisors were trained for 1 day on the data collection approach of the study. The questionnaire was translated into local languages, Amharic and Afan Oromo, by an expert and back-translated into English by another person to check for consistency. A pretest was conducted among 60 adults (5% of the sample size) living in Haramaya town, which is nearest to the study areas but not part of the study areas, to see the applicability of the instruments, and feedback was incorporated into the final tool to improve the quality. Supervision was done by the supervisors and principal investigator throughout the data collection period and checked daily for completeness and consistency of questioners.p-value less than 0.20 in bivariate analysis were entered into the multivariable logistic regression analysis. To estimate the strength of association between an outcome and explanatory variables, an adjusted odds ratio (AOR) with a 95% confidence interval was reported. A p-value of less than 0.05 is considered statistically significant. Multi-collinearity was assessed by the variance inflation factor (VIF) to see a correlation between independent variables and VIF were less than 5 for all variables, indicating that there was no problem of multicollinearity between the predictor variables. The goodness of model fitness was checked by using the Hosmer-Lemeshow test.The data were coded, cleaned, and entered into Epi Data version 3.2 and then exported to Stata version 16 for analysis. Bivariable and multivariable logistic regression analysis was performed to identify factors associated with a common mental disorder. All variables with a Ethical clearance was obtained from the Institutional Health Research Ethics Review Committee (reference number: IHRERC/043/2022) of the College of Health and Medical Sciences of Haramaya University. Then data collection was initiated after a letter of permission was obtained from Haramaya University College of Health and Medical Sciences for each district and Kebeles administrator. Official permission was secured from woreda and the Keble administrator. Also, informed, voluntary, written and signed consent was obtained from each participant after explaining the purpose and importance of the study before the interviews. Participants were informed about the aim of the study and the advantage of the study; confidentiality, as there was no risk of being participants, and they have full right to halt in the middle of the interview. Written informed consent was taken from each participant before data collection began. Confidentiality was maintained at all levels of the study through anonymous data collection. During data collection, the COVID-19 prevention protocol was kept.A total of 1,168 participants were included in the study with a response rate of 97.98%. The median age of respondents was 40, with an interquartile range  years. More than half of the participants, 57.87% (676) were males and around two-third, 64.47% (753) were married. Regarding the educational status of participants 30.65% (358) were able to read and write, the majority of participants 46.49% (543) were Muslim religion followers and nearly three-fourth, 72.43% (846) were urban residents as show in Most of the participants, 97.17% (1135) reported no family history of mental illness. Around half 46.15% (539) of respondents were lifetime substance users and 34.59% (404) were chewing Khat during the last 3 months. But only 1.3% (15) uses hashish/cannabis during the last 3 months. Out of all study respondents, around 27.65% (323) and 33.73% (394) had a comorbid medical illness and moderate social support, respectively, as shown in In this study, participants who scored more than 6 of the 20 questions of SRQ-20 were considered as having common mental disorders. Therefore, using this cut-off point (> 6 of SRQ-20), the prevalence of common mental disorders among adults living in the Harari regional state was 21.3% . The prevalence of common mental disorder was higher among female residents (24.79%) than male residents (18.78%), widowed (26.36%) than married (15.94%), among unemployed (38.26) than government employer (18.18), older age of ≥55 years (24.47%) than youngers of 15–24 years old (14.50%), current Alcohol users (29.27%) than non-users (20.38%), and among those who had family history of mental illness (45.45%) than who had not it (20.62%). The SRQ-20 responses (yes/No percentages) by the respondents was illustrated in p-value less than 0.05.In bivariate logistic regression analysis variables like being female, single, divorced, widowed family history of mental illness, current alcohol use in the last 3 months, students, unemployed, age greater or equal to 55 years were significantly associated with common mental disorders. However, in the multivariate logistic regression analysis variables like a female, single, divorced, widowed, family history of mental illness, unemployed, and age greater or equal to 55 years were statistically significantly associated with common mental disorders with a In this study, the odds of having CMD among respondents with were female were about 1.31 times higher as compared to participants those being male [AOR = 1.31 ], and the odds of having common mental disorders among participants who were widowed, single, and divorced were 1.79, 2.03 and 3.06 times higher as compared to respondents who were married with [AOR = 1.79 ],  respectively.The odds of having common mental disorders among respondents who had a family history of mental illness were 3.21 times higher as compared to respondents who had no family history of mental illness [AOR = 3.17 ] and odds of having common mental disorders among participants who were unemployed was 1.97 times higher as compared to participants who were government employers [AOR = 1.97 ]. Besides, the odds of having common mental disorders among participants who were age greater or equal to 55 years was 1.05 times higher as compared to respondents who were found within the age category of 15–24 [AOR = 1.10 ] as shown in Common mental disorder is a public health problems that cause impact to the individuals, their family and communities in both developed and developing countries . The gloHowever, the result of this study was lower than another community based study conducted in the United Kingdom among 8,191 residents using the General Health Questionnaire-12 items, 24.6% , in ChilOn the other hand, the finding of the current study was higher than the study done in rural Kenya among 50,000 population, using the Clinical Interview Schedule-Revised (CIS-R),10.8% , in soutIn this study, the odds of having common mental disorders were higher among female participants as compared to males. This result is supported by a study conducted in Kenya , South ARespondents who had a family history of mental illness were 3.17 times more likely to experience common mental disorders as compared to those who had no family history of mental illness. This finding was supported by a previous study carried out in Addis Ababa, Ethiopia . The posRegarding marital status, participants who were, widowed, single, and divorced were 1.79, 2.03, and 3.06 times more likely to experience common mental disorders, respectively, as compared to participants who were married. This result was supported by the study done in Kenya , and EdmWith respect to their occupational status, unemployed respondents were 1.97 times more likely to have common mental disorders as compared to governmental employed participants. This was supported by studies conducted in England , 40. TheThis study has some limitations. First, since the study design was cross-sectional, it cannot show the causal relationship. Second, during face-to-face interviewing the effect of the interviewer on the respondent can cause social desirability bias. Third, due to the nature of the data collection tool, recall bias can be considered a limitation. Fourth, the SRQ-20 is a screening instrument and not diagnostic of mental disorders. Fifth, the population studied were not screened for recent stressors, like loss to rule out bereavement as a confounder.The finding of this study showed that prevalence of common mental disorders was high in the community of Harari region state, Eastern Ethiopia. Being female, single, divorced, widowed, unemployed, having a family history of mental illness, and age were significantly associated with common mental disorders. Focusing on these identified associated factors, screening, early identification, and development of a program to prevent and provide appropriate home-based interventions for common mental illnesses in the community should be great attention to all stakeholders.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Health Research Ethics Review Committee (reference number: IHRERC/043/2022) of the College of Health and Medical Sciences of Haramaya University, eastern Ethiopia. The patients/participants provided their written informed consent to participate in this study.TA contributed to conceiving the original idea, designing and conducting the study, analyzing the data, preparing, critically reviewing, and approving the manuscript for publication. AB, HK, AA, KeB, KaB, FM, DD, FW, YA, ML, DW, TB, KG, SN, AN, GD, BB, JH, AE, AG, YD, AS, AH, HA, JD, and KN contributed to designing and conducting the study, analyzing the data, and reviewing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.