This paper thoroughly investigates material characterization, reliability evaluation, fabrication, and assembly processes of additively manufactured flexible packaging and reconfigurable on-package antenna arrays for next-generation 5G/mmWave wearable and conformal applications. The objective is to bridge the technology gap in current Flexible Hybrid Electronics (FHE) designs at mmWave frequencies and address the challenges of establishing future design standards for additively manufactured flexible packages and System-on-Package (SoP) integrated modules. Multiple 3D printed flexible materials have been characterized for their electrical and mechanical properties over the 5G/mmW frequency band (26–40 GHz), and the inkjet printed interconnects on 3D printed Polypropylene (PP) substrates demonstrated excellent electrical and mechanical performance during a 10,000-time cyclic bending test over typical wearable flexible radii down to 1 inch. A proof-of-concept flexible on-package phased array with an integrated microfluidic cooling channel on 3D printed substrates was fabricated and measured, demonstrating  This has created an increasing demand for future System-on-Package designs with complex functionalities in compact modules, which require real-time response, deployable packaging, and highly-integrated modules. These designs are essential for digital twins, structural health monitoring for megastructures, robotic applications with stringent  bending requirements such as those for Industry 4.0 applications and conformal intelligent metasurfaces. Flexible hybrid electronics utilizing flexible materials and rigid ICs are widely adopted in wearable designs due to their flexibility, but most of the FHE-based electronic systems are operating in low-frequency  because it is challenging to satisfy all the requirements for mmWave operation, including reliable performance within broad operational bandwidth, low parasitic losses from interconnects, and high gain phased array with beamforming abilities to overcome high path loss2. Additionally, these systems should be conformal in form factor with resilient and adaptable performance to bending and on-body effects from both RF and mechanical perspectives3, such as massively scalable MIMO enabling the implementation of antenna arrays with thousands of elements on virtually every conformal surface. However, most papers listing massive MIMO in the past lack practical deployment approaches.Next-generation data-driven Internet of Things (IoT) systems, large-scale smart cities, and wearable electronics for health monitoring are promised by the recent development of 5G/mmWave technologies with broadband operation and ultra-high data rates6, but the main limitation of these materials is that they cannot support customized packaging designs with complicated structures. On the other hand, the combination of 3D printing and inkjet printing has enabled the production of highly customized and intricate electronic devices with lower costs and less waste of materials. Previous research has extensively studied the electrical and mechanical properties of 3D printed and inkjet printed materials8, but very little attention has been given to the material characterization over the mmWave band. Although some past designs have demonstrated the 3D printed antennas, most of them focused on 3D printed rigid materials, such as horn antennas9 and dielectric lenses10; only a few reported flexible antennas with 3D printed substrates, including patch antennas below 5 GHz11 and dielectric reflectarrays at 28 GHz12. Recently, the first flexible mm-wave on-package antenna with an embedded energy harvester has been reported13, utilizing SLA printing and inkjet printing. The flexible module achieved 0.9 V output power at a 20 cm range with 59 dBm EIRP. Nonetheless, various challenges persist in implementing additively manufactured multi-chip modules on 3D printed flexible substrates. For example, conductive epoxy was used to attach a diode onto flexible substrates in13 because the 3D printed polymer substrates are sensitive to heat and cannot support thermal processing like soldering, and this poses a challenge for the accurate attachment of mmWave ICs due to their smaller pad and pin size. Heat dissipation of the IC may also become a problem with many flexible polymer substrates because their thermal conductivity is not as good as that of traditional rigid copper substrates like Rogers14. Another gap in past research is the lack of reliability evaluation of these flexible packages. Although all previous AM module applications refer to single bending-if any-, this is the first paper proposing 10,000+ bending reliable performance operability for practical bending radii for wearable and conformal 5G+ applications. The utilization of different additive manufacturing technologies has made FHE designs more accessible to in-house fabrication, but without generalized standards, it is difficult to evaluate the adaptability of the process and the quality of the designs.The solution proposed in this work is to utilize additive manufacturing (AM) techniques, including 3D printing and inkjet printing, to create on-demand customizable mmWave flexible packaging and reconfigurable on-package phased arrays for 5G wearable applications. A general demonstration of the proposed idea is shown in Fig. In this work, the aforementioned challenges and technology gaps have been successfully addressed. Different flexible materials from both Fused Deposition Modeling(FDM) printing and Stereolithography (SLA) printing were characterized for their electrical and mechanical properties over 26–40 GHz, covering the 5G mmWave frequency band. The fabrication process of inkjet printing onto 3D printed substrates has been optimized, and the fabricated microstrip line interconnect samples on 3D printed low-loss Polypropylene substrates have demonstrated less than 0.1 dB/mm insertion loss over 24–40 GHz, even under reliability evaluations through 10,000 times cyclic bending tests over a 1-inch bending radius. Finally, an on-package phased array with an integrated microfluidic cooling channel was designed, fabricated, and assembled through a low-temperature process, as a proof-of-concept demonstration for the proposed packaging concept. The implementation of a flexible phased array in this design allows additional reconfigurability that can be attached to any kind of surface while maintaining the antenna performance. The comprehensive investigation presented in this paper, including material characterization, fabrication process, reliability evaluation, and SoP implementation of additively manufactured 5G mmWave flexible packages, can provide guidelines for future FHE designs, and build the foundation of next-generation wearable technologies for various applications.15. Thus, it is necessary to create a comprehensive library of common 3D printed flexible materials for different applications. Insertion loss is one of the key performance parameters to evaluate substrate materials at mmWave frequencies because generating and maintaining signal power at mmWave frequencies is significantly more challenging than at lower frequencies, and minimizing insertion loss is crucial for ensuring high-quality and reliable signal transmission at mmWave frequencies16. The material properties of substrates, such as electric permittivity and surface roughness, can significantly impact the insertion loss. In particular, the real part of the permittivity is linked to dipole relaxation and lattice vibrations in the mm-wave range. The imaginary part of permittivity, known as loss tangent, corresponds to the ion friction effect that influences the loss properties of materials17. The typical value for loss tangent of a commercial substrate for mmWave applications is smaller than 0.01.Accurately characterizing the electrical properties of 3D printed flexible materials is the first step to designing flexible hybrid electronics systems. Even materials of the same type from different manufacturers can exhibit variations in their dielectric properties due to differences in fabrication methods and material composition18: Flexible 80A and Elastic 50A from Formlabs 3 (SLA printer); TPU 95A, Polypropylene, Soft PLA, and Ninjaflex TPU from Ultimaker S3 (FDM printer). They represent common types of commercially available 3D printed flexible materials. Transmission/reflection method19 was used to perform wideband characterization of the selected dielectric materials from 26–40 GHz. Samples of 3D printed materials were measured using WR28 waveguides and a Shockline 552B Vector Network Analyzer. To hold the samples, a waveguide sample holder of 2.99 mm thickness was used, and all materials under test were 3D printed as small cubes of size 20 method, which is the most straightforward algorithm in extracting dielectric properties by processing S parameter results. At least three samples were measured for each material, and the extracted properties were calculated in average values. Figure In this study, 6 different types of 3D printed flexible materials were considered21. FDM printers typically generate surfaces with uneven patterns, thus, accurate surface morphology characterization is necessary to evaluate the properties of different materials. It should be noted that surface roughness also depends on the printing setup, and the resulting surface quality can differ depending on the printing pattern22. All the FDM printed substrates used in this paper were printed with the typical zig-zag pattern. The highest surface roughness is measured along the perpendicular direction to the hatching pattern, and the resulting roughness values (Rq) are summarized in Table Surface roughness also needs to be taken into consideration when utilizing 3D printed materials for FHE designs. Since inkjet printing is commonly used as one of the metallization methods, the poor surface roughness of the substrates will lead to low print quality and high conductor loss23. The mechanical properties of the substrate material also affect the performance and reliability of the functional materials and components printed on top of it, such as conductive traces, sensors, and batteries. For example, if the substrate material is too rigid, it can cause cracking or delamination of the printed components, leading to the failure of the device.The mechanical properties of 3D printed flexible materials mainly influence the range of applications of FHE designs. For instance, in wearable devices, the substrate material needs to be flexible and stretchable to conform to the shape of the human body and allow for comfortable and continuous wear. It needs to be durable enough to withstand repeated bending, stretching, and twisting18, resulting in stress–strain curves. The curves were used to measure the elastic modulus, percent elongation at break, and tensile strength  of the materials. Additionally, a 2D digital imaging correlation (DIC) system  ink EMD573025 from Suntronic and Particle free silver (PFS) ink from Electroninks, which are commonly used conductive inks for metallization of different flexible substrates. The SNP ink was sintered at 130 26. The skin depth is the distance below the surface of a conductor where the current density has diminished to 1/e of its value at the surface. It is inversely proportional to the conductivity of the silver ink at certain frequency. At 30 GHz, SNP ink needs at least 2.205 um, while PFS ink needs to have at least 1.35 um. Considering the coffee ring effect of PFS ink after sintering, it would be difficult to uniformly satisfy the skin depth requirements in the cross-section, meaning the effective resistance will significantly increase, thus bringing in additional conductor loss. Additionally, the required sintering temperature of the selected PFS ink is much higher than that of the SNP ink, which could pose a challenge to many 3D printed polymer substrates that are highly sensitive to thermal treatment. A potential solution to this challenge is to use UV sintering instead of heat sintering to avoid any potential thermal damage to the substrate.In this study, an inkjet printer, Fuji Dimatix DMP-2850, and 10 pL cartridges were used to print conductive inks with a 20 um drop spacing for 8 layers onto a PET substrate for ink characterization18. Specifically, a 1 cm The nanoindentation technique was employed to obtain the elastic moduli of two inks27, as shown in Fig. 27, which is an epoxy-based photoresist that can be cured by a UV crosslinker, with a dielectric constant of 3.2 and a loss tangent of 0.04 at 24.5 GHz28. Figure Evaluation of the fabrication process is essential for the successful design and manufacturing of FHE devices. It is necessary to optimize the inkjet printing process onto 3D printed substrates, to understand the capabilities and limitations, and improve the reliability and performance of the final prototype. To evaluate the inkjet printability of conductive ink on different materials, SNP ink was utilized in this study. The printing quality of silver ink on 3D printed materials is mainly influenced by surface wetting and surface roughness. Surface wetting can be enhanced by employing UV Ozone treatment, which is an efficient and simple technique for increasing the hydrophilicity of plastic surfaces. The duration of UVO treatment may differ for various substrates. For instance, in the case of PP substrate, it has been found that 3 minutes of cleaning can lead to the best printing quality for SNP inkTo validate the electrical properties of 3D printed PP substrates and confirm the feasibility of the fabrication process, microstrip line samples were fabricated on 3D printed PP substrates using the inkjet printing process as mentioned above. Different metallization thicknesses of 3, 6, and 9 layers of silver ink were printed, and the average de-embedded insertion loss of different prototypes was shown in Fig. 18. Four sizes of cylindrical polycarbonate mandrels (outer radius: 1–4 inches) were used to sequentially bend microstrip lines wrapped around them, with the ink layer facing outward. S21 measurements were taken before and after bending, with the smallest mandrel inducing the largest insertion loss, as shown in Fig. To further investigate the reliability of microstrip lines with different metallization thicknesses (number of inkjet printed conductive layers) using the proposed fabrication method, monolithic bending tests and cyclic bending tests were conductedAn automated mandrel testing system was then designed for cyclic bending tests. The experiments involved taking S21 measurements before and after 1000, 5000, 7500, and 10,000 bending cycles for three sets of samples comprising 60 mm long microstrip lines with 3, 6, and 9 layers of printed silver ink. The results, presented in Fig. 2. Previous studies have demonstrated the fabrication of ramp interconnects through inkjet printing, aerosol jet printing, and 3D printing, and this paper is the first testing of the reliability of this type of interconnect up to 10,000 cycles. Two ramp interconnects were fabricated using SU-8 layers on both sides of a zero-ohm resistor and connected to 1.5 cm long microstrip lines that lead to two pads for resistance measurement, as shown in Fig. 27. These findings can guide the design and fabrication of reliable inkjet printed SNP-based electronic designs for wearable and conformal applications in 5G and B5G mmW frequencies.Similar cyclic bending tests were also performed on inkjet-printed ramp interconnects. As mentioned in previous sections, one of the key requirements of mmWave packaging is the realization of fully printed interconnects that feature ultra-low loss and “rugged” mechanical integrity, even for challenging bending radii. Compared to traditional interconnects such as wire and ribbon bonds that suffer from longer loop lengths and higher parasitics at mmWave frequencies, inkjet-printed ramp interconnects offer more robust, planar, and conformal structures with improved RF performance even in challenging configurations29. Figure 30, inkjet printed monopole phased array on 3D printed PP substrate, and 3D printed microfluidic channel. The antenna element in this work is a circular monopole antenna with back reflectors, to achieve wideband performance covering the entire 5G mmW frequency band from 24 to 40 GHz. Conventional UWB antenna elements like horn antennas and Vivaldi antennas are relatively bulky31, which become challenging in compact integration. Monopole elements can be planar and miniaturized, but they demonstrate omnidirectional or bi-directional radiation patterns, which are not preferred in many applications, such as wearable devices requiring good flexibility and isolated radiation from the human body. The design proposed in this paper overcomes these limitations in terms of size and radiation pattern, by incorporating a back reflector and a side reflector with the circular loop monopole elements, enabling broadside radiation with enhanced beamforming and reduced side lobe levels. The design guidelines for circular ring monopole antennas can be found in past research32. The beamformer IC can integrate multiple channels, and each has independent phase and amplitude control in their transmit and receive paths, allowing the RF and microwave front end to electronically steer the antenna beams for an optimum transmission aiming pattern. To match the 50 Ohm impedance from each RF output port of the IC, the width of the antenna feedlines is designed to be 0.5 mm, which is calculated based on the relative permittivity (2.34) and the thickness (0.2 mm) of the 3D printed PP substrate33. Additionally, all the feedlines are designed with the same electrical length, allowing the beamformer IC to achieve beam steering by adjusting the phase shift between elements. The antenna array was inkjet printed onto the PP substrate using the same process as explained in the previous section.To demonstrate the concept of smart packaging integration using the aforementioned additive manufacturing techniques, a 2.5D ultra-wideband (UWB) antenna array with embedded beamformer IC and the microfluidic cooling channel was fabricated on 3D printed Polypropylene substrate and Flexible 80A substrate34. However, for the FDM printed substrate used in this paper, the uneven surface pattern makes it impossible for ACA to reach the uniform pressure condition to generate joints . On the other hand, traditional methods using conductive epoxy have shown higher conductivity but limited reliability35. Therefore, the method in this paper, using conductive epoxy for attachment and applying ACA to enhance mechanical strength, has achieved a balance between electrical connection and mechanical reliability.Attaching the beamformer IC to the inkjet printed antenna array was the most challenging step during the assembly process due to the sensitivity of 3D printed polymer materials to thermal processes. Conductive epoxy is often employed in this regard, but its limitations include difficulty in effectively constraining the epoxy deposition within the pad area, which could cause a short between neighboring pins if there is a small misalignment of the IC. Additionally, the IC could detach easily when the epoxy dries up. An alternative solution is using Anisotropic Conductive Adhesive (ACA) material that is conductive only on the z-axis. The most recent research proved ACA materials to be very reliable and mechanically strongTo ensure accurate alignment of the IC pins, a stencil made from a transparent PET sheet was fabricated using a laser cutter. Conductive adhesive epoxy was applied to each pad of the inkjet printed IC footprint, and the IC was placed within the stencil. Uniform pressure was applied on top of the IC to ensure a solid electrical connection, and wires were attached to the DC pads for Serial Peripheral Interface (SPI) connection. Then an ACA material was used to seal the corners and edges of the IC to enhance mechanical connection. Vias were made through 0.2 mm diameter drilled holes filled with silver conductive paste, and the copper tape was used for ground plane metallization due to its quick fabrication and testing. The proposed approach can be readily applied to other flexible substrates, and it offers a practical solution for attaching components onto thermal-sensitive substrates.29. A 1.5 mm diameter was chosen to allow for maximum water flow while maintaining 3D print tolerances and flexibility, as illustrated in Fig. 36.As mentioned earlier, 3D printed polymer materials may suffer from poor thermal conductivity, which necessitates the integration of a microfluidic cooling channel for effective thermal management of the IC. In this work, the microfluidic channel was fabricated using Formlabs’ Flexible 80A material, printed as a cylindrical pipe with a diameter of 1.5 mm and walls that were 0.2 mm thick, and positioned underneath the IC’s center while attached below the antenna substrate layerThe measurements of the beamforming performance of the integrated phased array module were conducted in an anechoic chamber, as depicted in Fig. 37:The measurement results, as illustrated in Fig. 29  material from Ultimaker is a suitable candidate for mmWave flexible modules, with a low loss tangent of 0.001, an elastic modulus of 230 MPa, and a Poisson’s ratio of 0.40. Additionally, inkjet printed microstrip lines on 3D printed PP substrate were tested for their reliability and performance, and it was found that 6 layers of silver ink provide a good balance of electrical performance and mechanical reliability. Finally, the proposed low temp IC alignment and assembly process, along with the “smart” packaging topology used in the flexible on-package phased array with a microfluidic channel, lays the groundwork for the development of next-generation 5G mmWave broadband flexible hybrid electronics (FHE), flexible multi-chip module (MCM), and phased-array on-package modules for wearable and conformal smart skin, digital twin and massive MIMO applications.


The field of pharmacogenetics (PGx) holds great promise in advancing personalized medicine by adapting treatments based on individual genetic profiles. Despite its benefits, there are still economic, ethical and institutional barriers that hinder its implementation in our healthcare environment. A retrospective analysis approach of anonymized data sourced from electronic health records was performed, encompassing a diverse patient population and evaluating key parameters such as prescribing patterns and test results, to assess the impact of pharmacogenetic testing. A head-to-head comparison with previously published activity results within the same pharmacogenetic laboratory was also conducted to contrast the progress made after 10 years. The analysis revealed significant utilization of pharmacogenetic testing in daily clinical practice, with 1,145 pharmacogenetic tests performed over a 1-year period and showing a 35% growth rate increase over time. Of the 17 different medical departments that sought PGx tests, the Oncology department accounted for the highest number, representing 58.47% of all genotyped patients. A total of 1,000 PGx tests were requested for individuals susceptible to receive a dose modification based on genotype, and 76 individuals received a genotype-guided dose adjustment. This study presents a comprehensive descriptive analysis of real-world data obtained from a public tertiary hospital laboratory specialized in pharmacogenetic testing, and presents data that strongly endorse the integration of pharmacogenetic testing into everyday clinical practice. The LPUH Clinical Pharmacogenetics Unit was established in 2013 and its clinical protocols were developed by clinical pharmacologists and geneticists in collaboration with the clinical services that regularly request genetic tests . Most ofThe primary objective of this study is to highlight the activity, challenges, and advancements achieved by our Clinical Pharmacogenetic Unit over a 1-year period. In addition, our study aimed to conduct a head-to-head comparison of real-world data from previously published activity results within the same tertiary Pharmacogenetic Unit, spanning two different time periods: period 1 (January 2014 and December 2016) and period 2 (August 2021 and September 2022). Ultimately, this study aims to contribute to the expanding evidence base that supports the integration of PGx testing into routine clinical practice.TPMT-NUDT15, tacrolimus/CYP3A5, voriconazole/CYP2C19, siponimod/CYP2C9, irinotecan/UGT1A1 abacavir/HLA-B57:01 and fluoropyrimidines/DPYD. MTHFR genotype is also analyzed following an institutional internal protocol, but no dose recommendation is done due to the lack of genotype-based clinical guidelines available. RYR1 and CACNA1S genes are only analyzed in patients who have presented an event clinically compatible with malignant hyperthermia.We conducted a retrospective, single-center study to assess the impact of PGx testing during routine clinical practice at the LPUH Clinical Pharmacogenetics Unit. Established in 2013, it operates as a multidisciplinary entity, integrating the Clinical Pharmacology Department and the Genetics Department, both of which hold ISO 9001:2015 certification. Both departments play an essential role within the Unit; the geneticist is responsible of the technical, analytical and genetic data interpretation, whereas the clinical pharmacologist provides expertise on possible drug interactions, integrates patient’s clinical status with the obtained phenotype, and emits individualized treatment plans to be prescribed by the petitioner. It has implemented treatment adjustment protocols for the following preemptively genotyped drug-gene pairs: thiopurines/The majority of requests received can be classified into two groups: when a request is made for drugs for which the PGx testing is required before treatment prescription (preemptive genotyping in high-risk populations), petitions and samples are directly forwarded to the genetics department for sample processing and analysis. The final report with genetic data provided by the geneticist includes dose-adjustment recommendations provided by the clinical pharmacologist who takes into account the patient’s clinical background, individual interactions, and other factors is sent to the requesting service. When a medical case is referred where a PGx test could be deemed appropriate or a request is made for a drug without established clinical protocols, the Clinical Pharmacogenetics Unit’s clinical pharmacologist assesses and decides, by integrating available clinical information with up-to-date pharmacogenetic evidence, whether a PGx test is indicated. If the PGx test is recommended, the analysis is performed and a PGx report is generated. All PGx reports are performed according to the recommendations of the European Molecular Genetics Quality Network (EMQN) . An examThis study included patients with various pathologies with a PGx test performed in a local pharmacogenetic laboratory between August 2021 and September 2022. Demographic characteristics including age and gender as well as relevant future or current prescription, PGx data and diseases, were obtained from medical records and the laboratory information system over a 1-year period.A descriptive analysis of the study population was carried out which included the number of tests performed, what gene was requested, genotyping results and its proportion attending the total amount of requests of each type. Variables were described using the number of participants (n), mean and it is minimum and maximum range. We then compared the results obtained during from 2014 to 2016 with those obtained from 2021 to 2022.® technology ; PCR combined with electrophoresis (for the Pharmacogenetics of Irinotecan); Real-time PCR (Hypersensitivity to Abacavir); Next-Generation Sequencing .Blood samples were obtained in Vacutainer EDTA tube . DNA was isolated using a Chemagen extraction robot . Within our Hospital’s service portfolio, the technology used for each type of test was as follows: OpenArray® System  following manufacturer instructions. This panel simultaneously analyses 120 single nucleotide variants (SNVs) in 8 genes using TaqMan™ probes. Each TaqMan™ SNV assay contains two allele-specific probes and a primer pair to detect the specific SNV target. The validation study conducted for this technology was previously published  located in a TATAA consensus element in the UGT1A1 promoter, The forward primer sequence was GATTTGAGTATGAAATTCCAGCCAG, and the reverse primer sequence was CCAGTGGCTGCCATCCACT, which was fluorescently labeled with FAM. The PCR conditions consisted of an initial denaturation at 94°C for 3 min, followed by 31 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 7 min, and a final extension at 72°C for 7 min. Subsequently, fluorescent fragment sizing was performed using capillary gel electrophoresis. The analysis was carried out with GeneMapper® Software v4.0. The star allele was determined based on the length of the amplicon.Genotyping test for irinotecan pharmacogenetics study analyses by specific fluorophore-labeled PCR the most common genetic variant affecting HLA-B to establish presence or absence of *57:01 allele, associated hypersensitivity reactions to abacavir. The study was performed using the commercial GENVINSET® HLA B57 kit , which allows the detection of the HLA-B*57:01 allele using specific primers. The study was performed on the Cobas z 480 analyzer .The abacavir hypersensitivity study was conducted using genotyping test of RYR1 and CACNA1S genes by a massive sequencing panel (NGS) of our own design using the KAPA HyperChoice technology . Sequencing was performed on the Novaseq6000 or HiSeq4000 platform . Bioinformatics analysis was carried out by the Bioinformatics Unit of the genetics department. Variant classification was performed based on the recommendations of the American College of Medical Genetics and Genomics (https://varnomen.hgvs.org/). The project accession PRJEB66347 in European Nucleotide Archive (ENA) has the high throughput sequencing data from cases of this study.The malignant hyperthymia testing was performed by analyzing the coding regions of the Genomics  and variThis study was conducted under the approval of the ethics committee of the La Paz University Hospital (PI-2915) and in conformance with the principles of the Declaration of Helsinki.Since its establishment within LPUH in 2013, the Clinical Pharmacogenetics Unit has conducted genotyping for an approximate total of 5,000 patients. The overall hierarchical functioning of the Unit varies based on the type of request received and is summarized in By comparison, the total number of PGx tests carried out between January 2014 and December 2016  by this Previously reported data revealed that the Internal Medicine/Infectious Disease department accounted for 76.3%  of the requested actionable genetic marker tests. However, in the current study, among the 17 different medical departments that sought PGx tests, the Oncology department accounted for the highest number, representing 58.47% (383 out of 655) of all genotyped patient .DPYD  and UGT1A1  genetic testing was limited almost exclusively to Oncology. Dermatology was the main department that requested PGx combined tests for TPMT-NUDT15 . Both Dermatology and Paediatric Haemato-Oncology had an equal number of requests for PGx tests for MTHFR . Paediatric Haemato-Oncology had the highest percentage of requests for PGx tests for voriconazole . Paediatric Nephrology was the main department ordering CYP3A5 genotyping , while CYP2C9 genotyping was exclusively ordered by the Neurology department . The HIV Clinic of Internal Medicine was the sole department ordering HLA B57:01 genotyping.The department performing DPYD . The PGx tests performed for thiopurine S-methyltransferase (TPMT) and NUDT15 gene accounted for 17.82% of the total , followed by MTHFR at 5.68% , abacavir HLA B57:01 testing at 4.63% , CYP3A5 at 3.41% , and Voriconazole PGx (CYP2C19) at 3.32% . The least requested tests were the ones involving CYP2C9 at 1.22%  and malignant hyperthermia risk assessment, which accounted for only 0.70% of the total .Over a third of the 1,145 PGx tests  was consistently under 10 days across all instances. The shortest MRT was observed for the DPYD test (6.36 days), while the greatest MRT was seen for the CYP3A5 test (8.66 days).The mean response time (MRT) was determined as the number of elapsed days between the date of test request and the date of result report upload and exhibited a range of 1–176 days. The overall MRT for all tests was calculated to be 8.36 days, with a corresponding MRT for all PGx tests included in the OpenArrayOf the 655 individuals who underwent genetic testing, 83.21% (545/655) were performed prior to the initiation of treatment (preemptively). A total of 1,000 PGx tests were requested for individuals susceptible to receive a dose modification based on genotype. Among these cases, it was found that 7.60%  of the individuals received a dose adjustment according to the genotype results .DPYD, UGT1A1, CYP2C19, CYP3A5, and HLA-B57:01 indicated the following: 3.86% (15/388) of DPYD tests and 11.01% (37/336) of UGT1A1 tests required treatment adjustments of 5-Fluorouracil/Capecitabine and Irinotecan, respectively. A proportion of 13.15% (5/38) of CYP2C19 genetic tests prompted the administration of a different treatment from voriconazole. In the case of CYP3A5, 25.64% (10/39) of PGx tests led to adjustments in the starting dose of Tacrolimus in eligible kidney transplant patients. Finally, it was noted that 13.63% (9/66) of the TPMT/NUDT15 tests conducted resulted in a modification of the dose of either Azathioprine or 6-Mercaptopurine. Only 3 out of 14 CYP2C9 tests warranted dose modification of siponimod. In addition to pharmacogenomic tests performed using OpenArray technology, the results of HLA-B57:01 genotyping indicated that 11.32% (6/53) of patients were at elevated risk for developing abacavir hypersensitivity, thereby requiring an alternative Highly Active Antiretroviral Therapy (HAART) regimen  of individuals possessed at least one clinically relevant variant in the RYR1 gene. It is worth noting that all genetic assessments for malignant hyperthermia were performed on patients who had previously experienced a compatible adverse event in the operating room, hence none of these tests were conducted preemptively. One of the identified variants, NM_000540.2 (RYR1):c.15014C>T(p.Thr5005Met), was classified as a variant of uncertain significance, which indicates that further analysis, such as a more comprehensive assessment, would be required to draw definitive conclusions regarding the subject’s increased or decreased risk of developing malignant hyperthermia. On the other hand, the other detected variant, NM_000540.2 (RYR1):c.14545G>A p., was classified as a pathogenic variant.With respect to the Finally, The role of preemptive pharmacogenetics in enhancing treatment efficacy and significantly reducing adverse drug reactions is well established, as evidenced by recent publications . We haveOur analysis encompassed a total of 1,000 PGx tests specifically requested for individuals deemed susceptible to benefit from a dosage/treatment modification based on their genetic profile. The results revealed that a noticeable amount of these tests, 7.60% , led to modified treatments being implemented according to the genotype outcome. Few evidence has been published describing global prevalence of PGx variants in a similar population cohort to contrast this figure .DPYD of patients with decreased allele activity score (3.86%) differed from similar studies conducted in European cohorts that reported a higher prevalence, albeit in a larger sample size  highlighted P450 2C19 as their most frequently requested test, constituting 50.2% of their total inquiries  which has diminished by nearly 91% annually (corresponding to a net disparity of 506 petitions) when compared to our present findings. This unmistakably signifies a change in the management of HIV patients by healthcare professionals, wherein the utilization of abacavir has been relegated to a later stage when compared to the contemporary approach of HAART combinations  of capecitabine and 5-FU and security alerts issued by the Spanish Agency of Medicines and Medical Devices , something not uncommon given the increasing flux of patients across borders.TPMT in a patient that has been subjected to an orthotopic liver transplant, where the donor’s genotype can condition whether or not the patients suffer side effects in response to treatment  or to do so in cases that were incongruous: a petition for a PGx test for reatment .CYP3A5, for a kidney transplant recipient who was already undergoing treatment adjustment as per trough blood concentrations. In such a scenario, patient genotype would be of little clinical relevance, with blood concentrations guiding any treatment modifications (Likewise, it was frequently observed that petitioners would request a PGx test, such as ications .In our hospital, we have developed an internal record-keeping system that helps us identify instances of duplicate orders or cases where pharmacogenetic testing has been previously conducted for a particular patient. This internal system serves as a safeguard to prevent unnecessary repetition of tests. Additionally, for patients already undergoing therapeutic drug monitoring, such cases are identified through a manual review of the patient’s medical history by the clinician responsible for handling the test request. This review process also allows us to prioritize and manage cases that require urgent pharmacogenetic results.It therefore becomes apparent that genetic information is complex, and effectively translating it into actionable recommendations for clinicians requires clear guidelines and standardized reporting formats. Our Clinical Pharmacogenetics Unit exemplifies a model useful to bridge some of these challenges by integrating the interpretation of complex genetic information with the patient’s clinical status, therapeutic goals, and concomitant therapy. Something only possible by establishing a multidisciplinary unit that comprises expert geneticists and clinical pharmacologists with pharmacogenetic expertise.The findings of this study should be interpreted within the context of several limitations. Despite being active for 10 years, the data collected by the Clinical Pharmacogenetics Unit has been subject to various information system changes as the LPUH adapted its Information Technology infrastructure, and electronical medical health records platform among others. This precluded the study from spanning a longer time period, as collected data from the inception of the Unit was limited. The impact of the COVID-19 pandemic also hindered the activity of the Unit, not only because it suffered a complete cessation of activity during the lockdown period, but because in order to ease the economic burden caused by the pandemic financial reallocations were carried throughout the SNHS that rippled through units and departments that, despite being of great relevance for individual patient care, were not considered a priority at the time . The absThis study represents a significant contribution as it is, to the best of our knowledge, the first to offer comprehensive insights into the activity of a Clinical Pharmacogenetics Unit over two different time periods in the same setting. By doing so, we provide a deeper understanding of PGx practices in a real-world setting, with both their challenges and achievements. The findings presented highlight a notable rise in the utilization of PGx testing, indicating a growing awareness and acknowledgment among healthcare professionals regarding its significant role in customizing treatments according to individual genetic profiles. This increasing acceptance signals a shift towards a more personalized medicine, emphasizing the aim of delivering optimized and safe therapeutic interventions for every patient. By leveraging the insights provided by PGx testing, healthcare providers can make informed decisions and ensure the administration of treatments that are both efficacious and tailored to each patient’s unique genetic makeup.The real-world data presented in this study contributes to the growing body of evidence supporting the integration of PGx testing into daily clinical practice, allowing for tailored treatments based on individual genetic profiles, ultimately enhancing patient care. The implementation of PGx strategies that include a multidisciplinary approach that includes a geneticist and a clinical pharmacologist has the potential to significantly enhance the efficient utilization of available resources within healthcare systems, bringing us one step closer to a truly individualized healthcare. By optimizing the efficacy of current treatments and reducing the incidence of adverse events, pharmacogenetics can also contribute to a more sustainable healthcare system. However, further studies are needed to comprehensively explore and validate the full extent of these benefits, ensuring that the integration of pharmacogenetic and economic approaches is evidence-based and widely applicable.


Castanopsis kawakamii was examined. The aim of the study was to determine the optimal seed position to promote regeneration. Allocation strategies were well coordinated in the emerged seedlings from different seed positions. Seedlings from seeds positioned above litter layers of different thicknesses (~40 and 80 g of litter) allocated growth to leaf tissue at the expense of root tissue (lower root mass fraction) and increased nitrogen (N) and phosphorus (P) accumulation and nutrient use efficiency. Seedlings from seeds positioned beneath a deep litter layer allocated most growth to roots  to capture available resources at the expense of leaf growth. Seedlings from seeds positioned on the forest floor allocated most growth to roots to obtain limiting resources. Further, we also found that these traits were clustered into three groups based on trait similarity, and the cumulative interpretation rate was 74.2%. Thus, seed relative positions significantly affected seedling growth by altering the allocation of resources to different organs. The different strategies indicated that root N:P ratios (entropy weight vector was 0.078) and P nutrient use efficiency were essential factors affecting seedling growth in the subtropical forest. Of the seed positions analyzed, beneath a moderate litter layer (~40 g of litter) was the most suitable position for the growth and survival of Castanopsis seedlings. In future studies, field and lab experiments will be combined to reveal the mechanisms underlying forest regeneration.Plants allocate growth to different organs as a strategy to obtain limiting resources in different environments. Tree seeds that fall from a mother tree settle on, within, or below the forest floor and litter layer, and their relative positions can determine seedling biomass and nutrient allocation and ultimately affect survival to the sapling stage. However, how emerged seedling biomass and nutrients of each organ are affected by seeds in different positions is not yet completely understood in subtropical forests. Therefore, an experiment was conducted with seeds positioned above the litter layers of different thicknesses, on the forest floor, and beneath the litter layer, and the influences of seed position on biomass allocation and nutrient use efficiency of emerged seedlings of  For example, plants allocate growth to shoots or roots to, respectively, capture light or soil water and nutrients, which indirectly affects plant regeneration . In foreLitter or soil is the initial physical environment that seeds contact after falling from a tree , and seeCastanopsis is a common tree genus in subtropical evergreen broad-leaved forests and is widely used in forest restoration . The region has a subtropical monsoon climate (C. kawakamii (average age > 100 years), Litsea subcoriacea, and Schima superba , full and intact seeds were collected and then stored in sand. Before conducting a germination experiment, we placed seeds in distilled water for 3 h, removed the floating and inferior seeds, and selected the intact and pest-free seeds for disinfection using 0.5% KMnOC. kawakamii natural forest prevented seeds from always contacting the forest floor. Thus, soil was collected from a depth of 10–40 cm in each sample plot. The soil from each plot was a mixture of soil from the east, south, west, north, and center positions of each plot. The soil was passed through a 5-mm sieve to remove roots and stones. The soil was then sterilized in an autoclave for 30 min at 120°C to reduce the probability of seeds in the soil being infected by pathogens (The relatively thick humus layer (0–10 cm) and intricate plant roots systems on the forest floor of the athogens .2.3C. kawakamii natural forest (18%–27%) (C. kawakamii (432.5–922.5 g/m2).A germination experiment was conducted in a large indoor laboratory in January 2019. Germination pots measured 38.5 cm × 27.5 cm × 14 cm, and litter weight was calculated according to pot volume. A completely randomized experimental design included seven seed-position treatments. Seeds positioned above the litter layers included one positioned in a 2 cm thick litter layer (~40 g of litter) and one positioned in a 4 cm thick litter layer (~80 g of litter). That is, seeds were in direct contact with the litter layer. Seeds’ relative positions on the forest floor or beneath the litter layer meant direct contact between the seeds and forest soil. Seeds positioned on the forest floor without litter cover layer. Seeds positioned beneath the litter layer meant that they are covered by 2 cm (~40 g of litter), 4 cm (~80 g of litter), 6 cm (~120 g of litter), and 8 cm (~160 g of litter) of litter . Each po18%–27%) , and lit2.4n = 3) and was regarded as one available value.In December 2019, functional traits were measured on individual seedlings. Three surviving individual seedlings in each experimental pot were harvested to measure biomass and nutrients. Fully expanded leaves of seedlings without obvious pests and diseases were selected, and roots were separated from the main stems and leaves. Each organ was carefully cleaned of soil with deionized water, ensuring the integrity of roots as much as possible. Plants’ tissues were placed in marked envelopes and returned to the laboratory for further analysis. Roots, stems, and leaves were oven-dried to constant weight at 65°C for 72 h, and dry matter mass (0.0001 g) was determined to calculate traits related to biomass allocation. Root: shoot ratio was calculated as root biomass divided by the sum of stem and leaf biomass. Mass partitioning into photosynthetic and non-photosynthetic tissues was calculated as photosynthetic tissue (leaf biomass) divided by non-photosynthetic tissue (sum of stem and root biomass) . Total p2.5Dried seedling roots, stems, and leaves were crushed by mortar and sieved through a 0.15-mm mesh. Total N contents of roots, stems, and leaves were determined by an elemental analyzer , and total P contents were determined by inductively coupled plasma emission spectrometer . The N:P ratios in root, stem, and leaf were calculated as N contents divided by P contents of the different tissues. The total N and P contents of plants were determined by biomass-weighted mean values of N and P contents of leaves, stems, and roots. Total N and P accumulation were determined by multiplying total plant biomass and total nutrient contents. Plant nutrient use efficiency was calculated as total plant biomass divided by total plant nutrient content .2.6To analyze differences in biomass and nutrient allocation of emerged seedlings from different seed positions, the normality of all variables was first tested using the “shapiro.test” function. Then, “aov” and “duncan.test” functions in the “agricolae” package were used to identify significant differences among means of seedling traits from different seed positions . To asseC. kawakamii seedlings, biomass and nutrient contents were incorporated into a principal component analysis (PCA) using the “prcomp” function from the “factoextra” package, and the first two dimensions were extracted  of seedlings from seeds positioned on the forest floor  is consistent with the water limitation theory . When su4.2Plant N and P contents and N:P ratios reflect the dynamic balance of nutrients and adaptation strategies of plants as affected by resource supply and demand , and areThe N:P ratio is used to evaluate nutrient-limiting conditions in plants, and N:P ratio < 14, 14 < N:P ratios < 16, and N:P ratios >16 generally indicate limitations of N, both N and P, and P, respectively . In this4.3Nutrient accumulation and use efficiency are important factors affecting plant regeneration and adaptation strategies . A moder4.4In this study, relationships were explored between different positions of seeds and biomass and nutrient contents of seedlings of a dominant tree species to better understand how seed position affects regeneration in subtropical natural forests S2. AccoRoot, stem, and leaf N:P ratios <14, as well as relatively high leaf N:P ratios and relatively low coefficients of variation . A compr5To investigate factors affecting seedling regeneration in subtropical forests, this study explored the effects of different seed positions on above- and belowground biomass and nutrient contents of emerged seedlings of a dominant tree species. One critical finding was that biomass allocation in different organs was well coordinated. Emerged seedlings from seeds positioned above litter and beneath a moderate litter layer (~40 and 80 g of litter) had higher leaf allocation at the expense of root and stem tissues than seedlings from other positions. Emerged seedlings from seeds positioned beneath a deep litter layer (~120 and 160 g of litter) had higher root allocation at the expense of leaf tissue than seedlings from other positions, which was necessary to break through the physical barrier of litter. Biomass and nutrient allocation of emerged seedlings from seeds positioned on the forest floor indicated a decrease in investment in shoot tissue to achieve growth. Traits of biomass and nutrient were also comprehensively analyzed, and the analysis indicated that seedlings from seeds positioned beneath a moderate litter layer (~40 g of litter) facilitated the regeneration of a dominant tree species in the natural community, because those seedlings had relatively high N and P accumulation and nutrient use efficiency (N:P ratios < 14). Thus, protective mechanisms that occur in seeds positioned beneath a litter layer are particularly important for the natural regeneration of seedlings. In the future, seedling regeneration in subtropical natural forests could be promoted by adjusting litter thickness or adding N elements.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JZ: conceptualization, formal analysis, investigation, methodology, writing–original draft, writing–review, and editing. LJ: investigation and formal analysis. LC: writing–review and editing. XJ: investigation. CX: investigation. JL: conceptualization, supervision, writing–review, editing, and funding acquisition. YY: writing–review and editing. ZH: conceptualization, supervision, writing–review, editing, and funding acquisition. All authors contributed to the article and approved the submitted version.


Piriformospora indica) on improving drought tolerance in moldavian balm , a medicinal and aromatic plant. For this purpose, a greenhouse study was conducted in a factorial experiment based on a randomized complete design with three replications. Results indicate that water stress reduces the membrane stability index (MSI), total chlorophyll content (Chlt), carotenoids, and maximum photochemical efficiency of photosystem II (Fv′/Fm′) in moldavian balm plants, while increasing superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) content compared to the control (no water stress). Inoculation with PGPBs and Piriformospora indica helped alleviate the negative effects of water stress. The highest MSI (48%) and Fv′/Fm′ value (0.82) were observed when inoculated with Enterobacter and Piriformospora, respectively, under non-water-stressed conditions. Inoculation with Agrobacterium, Piriformospora, and Enterobacter improved the Chlt and leaf proline contents, as well as the SOD activity under high water stress, compared to the non-inoculated control values. Furthermore, inoculation with Pseudomonas under high water deficit stress levels increased the MDA content (0.51 mmol g−1 FW) and H2O2 levels (0.40 mmol g−1 FW). The highest yield of flowering branches (2.414 g pot−1) in moldavian balm was obtained with Enterobacter. Based on the enhanced physiological and biochemical responses, as well as increased antioxidant enzyme activity that improve water tolerance in this plant, it is recommended to use PGPBs and Piriformospora indica fertilization.Plant growth-promoting bacteria (PGPBs) play a crucial role in mitigating the oxidative damage caused by water stress in different plant species. The aim of this study was to determine the effects of PGPBs and mycorrhiza-like fungi ( Dracocephalum moldavica L.) is an annual herbaceous plant that belongs to the Lamiaceae family. It is known for its production of essential oils and its medicinal-aromatic properties. The moldavian balm is native to the temperate climate of Asia but has been naturalized in Eastern and Central Europe, North Africa, China, and the northeastern United States. This plant is distributed in the northwest of Iran, including Tabriz, Urmia, Mazandaran, and the Alborz mountain ranges1. This annual plant is 25–75 cm tall and has numerous stems and blue flowers arranged in pseudo-whorls, growing in leaf axils. The essential oil of moldavian balm accumulates in the leaves, inflorescence, and stems in oil-containing cells1. Moldavian balm's essential oil is used for food, cosmetics, flavorings, and pharmaceutical purposes. The aerial part of moldavian balm is used in Iran for its stomachic, digestive, sedative, diaphoretic, and medicinal properties.Moldavian balm . A significant reduction in chlorophyll content can be observed due to water stress3. The osmotic adjustment, mainly through proline accumulation, is a typical response of crops to environmental stresses4. This decreases the osmotic potential and maintains cell turgor pressure at a high level, enabling plants to sustain their physiological processes. The reactive oxygen species (ROS) level can also increase in plants due to water deficit stress. ROS can disrupt normal cellular activities when it reaches the threshold level, potentially leading to increased membrane lipid peroxidation5. Then, the breakdown of fatty acids could result in the formation of small hydrocarbon fragments, including malondialdehyde (MDA)6.Water deficit stress reduces the availability and uptake of nutrients, which may lead to a decrease in transpiration, stomatal conductance, photosynthetic rate, and growth6. Helal and Samir7 found that modulating the activities of these enzymes can play a critical role in imparting plant resistance against water deficit stress. In medicinal plants, water deficit stress can also reduce solute and antioxidant accumulation8. Solinas et al.9 reported that water deficit stress is a significant factor that affects the synthesis of natural products, thereby altering the secondary metabolites in plants.Antioxidant enzymes, such as superoxide dismutase (SOD) and peroxidase (POD), help plants protect cellular and sub-cellular systems from the cytotoxic effects of active oxygen radicals10. Under water deficit stress conditions, inoculation with Piriformospora indica enhanced the activity of antioxidant enzymes, increased the levels of osmolytes, and protected the chlorophyll pigments11. In cropping systems, inoculation with Piriformospora indica (a mycorrhiza-like fungus)12 and PGPBs13 is a suitable strategy to mitigate the effects of water deficit stress. Inoculation with PGPBs improved root elongation, reduced the effects of water deficit stress under field and greenhouse conditions, enhanced nutrient mobility, and ultimately improved crop growth and production14. Inoculation with arbuscular mycorrhizal fungi (Glomus versiforme) improved the growth and herbage yield of moldavian balm under water stress15. The yield of Moldavian balm herbage decreased by 45% under high water deficit stress compared to the no stress treatment16.Plant physiology is directly affected by certain bacterial strains, which can increase the synthesis of plant hormones. In various crops, plant growth-promoting bacteria (PGPBs) play a crucial role in mitigating oxidative damage caused by abiotic stress by manipulating antioxidant enzymesPiriformospora indica can enhance certain physiological parameters, the activity of antioxidant enzymes, and the production of secondary metabolites in moldavian balm11. In addition, their use leads to a reduction in the use of chemical fertilizers in agroecosystems. Based on the documentation, there is no available information regarding the impact of PGPBs and Piriformospora indica on mitigating the adverse effects of water stress in this species. Therefore, this study was conducted for the first time with the aim of evaluating the effect of these compounds on specific biochemical and physiological attributes that contribute to drought tolerance and adaptation in moldavian balm.Using PGPBs and Agrobacterium sp., Pseudomonas fluorescens, Piriformospora indica and Enterobacter cloacae.The study was conducted twice, once in 2018 and again in 2019, in a research greenhouse at the University of Tabriz in Iran. The total duration of the experiment, from sowing to harvesting, was 92 days in both years. The two-factor factorial experiment was arranged as randomized complete block design (RCBD) with three replications. The factors were water deficit stress at four levels based on field capacity (FC) including no water deficit stress (85–100% FC), low water deficit stress (70–85% FC), medium water deficit stress (55–70% FC), high water deficit stress (40–55% FC) and inoculation levels consisted of control (non-inoculated), inoculation with Agrobacterium sp. 14A-4, Enterobacter S16-3 and Pseudomnas sp. C16-2O were prepared in the soil biology laboratory, University of Tabriz, Iran. The bacterial strains (an overnight culture in 16 h) prepared in nutrient broth (NB) and after reaching the desired optical density was applied in the study. In the pot experiment the bagasse (sterilized): perlite (50:50) was used as a carrier. The mixture's moisture reached to 20% by adding distilled water, and then bacterial suspension (10 ml) containing 108 Colony-Forming Unit (CFU) ml−1 was added to the 100 g bagasse: perlite mixture and applied as inoculum. The NB medium contained beef extract 10 g, NaCl 5 g L−1, and peptone 10 g17.The bacterial strains consisted of Piriformospora indica fungi was provided from the collection of Soil Biology Laboratory, University of Tabriz, Iran. The Piriformospora indica was propagated on a plate in Kafer medium for 2 weeks under 24 °C17. After propagation, the mycelium of Piriformospora was added to the sterilized bagasse:perlite (50:50) carrier and applied as inoculum treatment in the experiment. The prepared inoculum was contained mycelium and of 104 spores g−1 of Piriformospora indica.The Agrobacterium, Pseudomonas, Piriformospora indica, and Enterobacter were added to the soil of the pots before moldavian balm sowing at rates of 8.0, 8.0, 10.0 and 8.0 g pot−1, respectively. After inoculation with Piriformospora indica and bacterial strains the soil surface in the pots was fog sprayed. No inoculum was used in control.The plastic pots with 25 cm diameter and 30 cm height containing 6 kg soil were used for the experiment. The pot's soil was provided from the University of Tabriz research field, which was sampled at a depth of 0–20 cm. The physiochemical characteristics of the soil are presented in Table Seeds of moldavian balm were purchased from Pakan-Bazr Isfahan, Iran. In all pots, ten seeds of moldavian balm were sown at the 1-cm soil depth and after seedling emergence and establishment, five plants were kept in each pot and the others were removed. Three pots were considered for all treatments. The moldavian balm pots were placed in a greenhouse with a relative humidity of 60–70% and a temperature of 22 ± 2 °C. The FC of the soil was determined using a pressure plate apparatus. The irrigation of pots was done by the weighing method based on water deficit stress levels to reach the desired FC level as needed. Water deficit stress levels were exerted from the 4–5-leaf stage of moldavian balm seedlings to the maturity of plants.18:At 50% flowering stage, water stress injury to the cell membrane was measured by electrolyte leakage (EL) using an electrical conductivity meter . The membrane stability index (MSI) was calculated using Eq. 18:1\docu19, Chlt contents and carotenoid contents based on Arnon20, Maximum photochemical efficiency of photosystem II (Fv′/Fm′) according to the Maxwell and Johnson21, water soluble carbohydrates (WSCs) using the method of Kochert22, POD as proposed by Ghanati et al.23, SOD as reported by Zhang et al.24, leaf proline (LP) content according to the Bates et al.25, MDA content as mentioned by Li26 and H2O2 was measured according to Alexiva et al.27. Leaf soluble protein (SP) content was also evaluated by the method of Bradford28. A sample of the plants (five) in each pot was taken to measure the mentioned attributes. To measure flowering branches’ yield, 25 cm above the branches were cut and their weight was recorded after they dried.RWC according to Teulat et al.The analysis of variance (ANOVA) was done as a two-factor factorial experiment (4 × 5) based on RCBD with three replications by SAS software ver. 9.4 . The experiment was carried out twice and the data of both experiments were pooled for analysis because the time × treatment interaction was not significant. The data of moldavian balm physiological and biochemical traits were used in ANOVA passed the tests of normality and homogeneity of variance. The Duncan multiple range test was used for mean comparison at P ≤ 0.05.The authors confirm that the use of plants in the present study complies with international, national, and institutional guidelines.Enterobacter in control conditions (85–100% FC)  . The highest MSI (48%) was observed in the inoculation with . The higAgrobacterium, Pseudomonas, Piriformospora, Enterobacter and control, respectively. Inoculation with Agrobacterium, Piriformospora and Enterobacter increased the moldavian balm Chlt content under all water deficit stress levels. Pseudomonas inoculation had no significant effect on Chlt content of moldavian balm. Under no water deficit stress level, inoculation with Agrobacterium, Piriformospora, and Enterobacter enhanced the moldavian balm Chlt content by 24.26%, 22.97%, and 19.19%, respectively, compared with control. Also under high water deficit stress level, inoculation with Agrobacterium, Piriformospora, and Enterobacter enhanced the moldavian balm Chlt content by 26.49%, 22.37%, and 12.59%, respectively, in comparison with control  was obtained under no water deficit stress level and inoculation with Piriformospora and Agrobacterium (2.35 mg g−1 FW). Generally, inoculation with Agrobacterium had the highest Chlt content under all water deficit stress levels compared with those in other inoculation levels. There was no significant difference among the inoculation with Piriformospora and Agrobacterium under all water deficit stress levels.Based on ANOVA results, the interaction of water deficit stress and inoculation was significant on Chlt content P ≤ 0.01). The mean comparison  was obtained in the inoculation with Agrobacterium in control conditions (85–100% FC). At this stress level, no significant difference was observed between inoculation with Agrobacterium and Piriformospora. The lowest carotenoid content (0.76 mg g−1 FW) was observed in the inoculation with Pseudomonas under high water deficit stress (40–55% FC). However, no significant difference was observed between inoculation with Pseudomonas, Piriformospora, Enterobacter, and non-inoculated control at this stress level . As shown in Fig. . As showPiriformospora in conditions without water stress. However, there was no significant difference between inoculation with this treatment and other treatments at this stress level. The lowest Fv′/Fm′ value (0.44) was observed in the inoculation with Pseudomonas under high water deficit stress (40–55% FC). Furthermore, no significant difference was observed between inoculation with Pseudomonas and non-inoculated control in this stress level.The interaction of water deficit stress with inoculation significantly affected Fv′/Fm′ value in moldavian balm (P ≤ 0.05). Data presented in Fig. −1 DW) in inoculated with Piriformospora under high water stress (40–55% FC) and its lowest (1.42 mg g−1 DW) in the non-inoculated control and the condition without stress (85–100% FC) were observed . The highest WSCs  in comparison with that in control  was observed in inoculation with Piriformospora, Agrobacterium, and Enterobacter under high water deficit stress levels. However, moldavian balm LP content was not affected by inoculation with Pseudomonas.The mean comparison of interaction indicated that in control, increasing the water deficit stress level from no to high water deficit stress level increased the LP content in moldavian balm significantly Fig. b. In Entrol Fig. b. The grPiriformospora and Pseudomonas had the most significant and lowest RWC (64.78% and 48.84%), respectively . However, the interaction of water deficit stress with inoculation did not affect RWC. The mean comparison results indicated that the no water deficit stress had the highest RWC (71.56%) and the lowest one was observed under a high water deficit stress level (46.7%). Inoculation with ly Table . The RWC−1 FW), and the lowest one (0.3159 μg−1 FW) was observed under no water deficit stress level (Table −1 FW) was observed in Enterobacter treatment that was not significantly different with those in inoculation with Agrobacterium and Piriformospora. The lowest POD activity (0.2146 μg−1 FW) was observed in inoculation with Pseudomonas which had no significant difference with that in control , respectively. The SOD activity was not affected by inoculation with Pseudomonas, while under all water deficit stress levels, inoculation of Agrobacterium, Piriformospora and Enterobacter increased the SOD activity. Inoculation with Agrobacterium, Piriformospora and Enterobacter increased the SOD activity by 53.63%, 46.0%, and 42.04%, compared with that in control, under high water deficit stress  was observed in inoculation with Agrobacterium under high water deficit stress. The lowest SOD activities were observed in inoculation with Pseudomonas which had no significant difference from those in control.Interaction of water deficit stress with inoculation significantly affected SOD activity in moldavian balm (P ≤ 0.01). Results indicate that in all inoculation treatments, the activity of SOD increased as the level of water deficit stress increased Fig. a. The in−1 FW) was observed in inoculation with Pseudomonas under high water deficit stress level that was not significantly different with that in control under high water deficit stress level. Inoculation with Pseudomonas under high water deficit stress resulted in an 80% increase in MDA levels compared to non-inoculated plants without water stress.The results of mean comparison for interaction showed that in all inoculation treatments the moldavian balm MDA content increased with increasing the water deficit stress level Fig. b. The hi2O2 content  was observed in inoculation with Pseudomonas under high water deficit stress level which was not significantly different from control (non-inoculated) under high water deficit stress level , and the lowest was obtained under no water deficit stress (0.71 mg g−1 DW). Inoculation with Agrobacterium, Enterobacter, and Piriformospora had the highest SP content  . However, the interaction of water deficit stress with inoculation did not affect SP content. The mean comparison results indicated that the high water deficit stress had the highest SP content (1.19 mg gy) Table .−1) was obtained in Enterobacter, which was not significantly different from those in inoculation with Agrobacterium and Piriformospora , and the lowest one (1.201 g.pot−1) was observed under high water deficit stress  and inoculation treatment (P ≤ 0.05) significantly affected flowering branches yield. However, the interaction of water deficit stress with inoculation did not affect flowering branches yield. Among the inoculation treatments, the highest FBY  increased the total chlorophyll content of the leaves by 30% compared to the non-inoculated treatment. In another experiment, it has been reported that inoculating Solanum melongena L. cultivars with Piriformospora indica reduced the adverse effects of drought stress on total chlorophyll levels30.MSI is considered one of the important selection criteria for assessing plant drought tolerance. Membrane lipids underwent significant changes in moldavian balm when it experienced water stress Fig. a. Subseqion Fig. b,c. In tPiriformospora had a higher Fv′/Fm′ value than the other treatments  with Piriformospora indica and Azospirillum improved the proline content and plant growth.Improved WSCs in plants treated with PGPBs and own Fig. a. Probabnas Fig. b. Under 32 in moldavian balm and Kazeminasab et al.33 in lemon balm . Inoculation with Piriformospora, Agrobacterium, and Enterobacter increased the RWC of moldavian balm. Hosseini et al.34 also observed that wheat plants colonized with Piriformospora indica had higher RWC under stress conditions. Similarly, Ahmad et al.35 reported that inoculation with Enterobacter did not affect the RWC of maize (Zea mays L.).The RWC of moldavian balm was significantly reduced by increasing the level of water deficit stress. Similar results have also been reported by Alaei et al.36 also reported that in rice (Oryza sativa L.), the activity of POD increased under water stress conditions. Increasing the activity of POD can be attributed to the excessive accumulation of ROS in plant cells under conditions of water deficit stress. Inoculation with Agrobacterium, Piriformospora, and Enterobacter increased the POD activity of moldavian balm compared to that of the control. Saravanakumar et al.37 also reported that in green gram (Vigna radiata L.), the activities of POD in plants inoculated with Bacillus subtilis and Pseudomonas fluorescens increased significantly compared to non-inoculated plants. Furthermore, inoculation with Dracocephalum moldavica with Micrococcus yunnanensis (PGPB) and Claroideoglomus etunicatum  improved the antioxidant activity, including POD enzyme, which can mitigate the detrimental effects of water deficit stress38.The POD activity was significantly enhanced by increasing the level of water deficit stress. Gusain et al.Agrobacterium resulted in the highest SOD activity among all the inoculation treatments under different water deficit stress levels  with Agrobacterium tumefaciens. El-Tayeb41 observed that the Phaseolus acutifolius plants that were inoculated showed a decrease in MDA content compared to the control plants (Vicia faba L.). This decrease in MDA content led to an increase in resistance to prolonged water deficit stress.The MDA content increased in all inoculation treatments, except for the 2O2 content increased significantly in all inoculation treatments as the level of water deficit stress increased. Inoculation with Pseudomonas resulted in higher H2O2 content under all levels of water deficit stress compared to the other inoculation treatments. Generally, inoculation with Pseudomonas had no significant effect on the reduction of H2O2 accumulation under water deficit stress conditions. Inoculation treatments, such as Agrobacterium, Piriformospora, and Enterobacter, significantly reduced the H2O2 contents under all levels of water deficit stress. Gusain et al.36 also reported that the H2O2 content increased under high water deficit stress levels, and the inoculation of rice with PGPBs reduced the accumulation of H2O2. Inoculation with PGPBs led to a reduction in ROS levels under water deficit stress conditions. Therefore, these microorganisms prevent the harmful effects of H2O2 on plant growth and development under conditions of water deficit stress.The H42. In this way, the increase in protein content resulting from stress and inoculation with these compounds has led to the regulation of plant osmosis. Nadeem et al.43 reported that inoculation with Agrobacterium under stress conditions helps to preserve soluble leaf proteins in crop plants. The use of Enterobacter has likely resulted in an increase in the yield of flowering branches by enhancing plant tolerance mechanisms to drought, compared to other treatments. Therefore, it is recommended to use Enterobacter to alleviate the detrimental effects of water stress in moldavian balm. Similarly, a study on Calendula  showed that the application of Enterobacter increased the number of flowering branches by providing nitrogen and phosphorus44. In general, although the physiological and biochemical response of moldavian balm to the application of the mentioned compounds varied, the positive impact of these compounds on the plant's ability to tolerate water stress was evident. Therefore, if other positive aspects of this species yield favorable results, it is recommended to promote its cultivation in arid and semi-arid regions worldwide, using similar techniques.One of the defense mechanisms during osmotic stress is the synthesis of proteins. Accumulation of ROS in the cell leads to the activation of specific signaling pathways that regulate gene expression, modifications in protein synthesis and activity, and ultimately prepares the cell to adapt to new conditionsPiriformospora indica under water stress. PGPBs, particularly Enterobacter, and Piriformospora indica, improved drought tolerance in moldavian balm. This was demonstrated by significant increases in physiological responses and the yield of flowering branches under water stress. The application of these compounds improves the activity and levels of enzymatic and non-enzymatic antioxidants, preserves the integrity of the cell membrane, and enhances the efficiency of photosystem II during water stress. This leads to a reduction in the adverse effects of osmotic stress in moldavian balm. Considering the environmental advantages of the mentioned compounds, such as their non-toxicity, natural origin, and biodegradability, they can be considered as suitable alternatives to chemical fertilizers in sustainable agroecosystems. Further research and experimentation are needed to fully explore the potential benefits and mechanisms of inoculating moldavian balm with mycorrhizal fungi and bacteria under water stress conditions. However, these approaches show promise in mitigating the adverse effects of water stress and improving the overall health and productivity of plants.Considering the global challenge of drought, the utilization of compounds that enhance plants' ability to tolerate water stress will play a crucial role in water management. For the first time, this study revealed the tolerance mechanisms of moldavian balm induced by PGPBs and


The improved antioxidant activity of entrapped resveratrol was evaluated on keratinocyte cells as a function of its concentration. Finally, to really propose the resveratrol-loaded LCNs for topical use, the systems were gelled by using two different gelling agents, poloxamer P407 and carboxymethyl cellulose, to improve the contact time between skin and formulation. The rheological features of obtained gels were evaluated using two important methods (microrheology at rest and dynamic rheology), before testing their safety profile on human healthy volunteers. The obtained results showed the ability of LCNs to improve antioxidant activity of RSV and the gelled LCNs showed good rheological profiles. In conclusion, the results confirmed the potentiality of gelled resveratrol-loaded nanosystems for skin disease, mainly related to their antioxidant effects.As many natural origin antioxidants, resveratrol is characterized by non-suitable physicochemical properties for its topical application. To allow its benefits to manifest on human skin, resveratrol has been entrapped within liquid crystal nanocarriers (LCNs) made up of glyceryl monooleate, a penetration enhancer, and DSPE-PEG 750. The nanosystems have been more deeply characterized by using dynamic light scattering and Turbiscan Lab The topical application of drugs has emerged as an increasingly favored solution, offering a promising alternative to other administration routes for addressing various medical challenges, particularly when a local effect of a drug is desired. Indeed, a topically applied drug is less involved in causing systemic side effects and it is not subjected to the first-pass effect that should induce a marked reduction in available drug dose . UnfortuThanks to the most modern technologies and discoveries, it has been possible to improve the skin-specific activity of drugs, permitting an efficacious topical administration.Several nanosystems were designed with the aim to lead the entrapped drug through skin structures, reaching the effective dose at its action sites. Examples include: ethosomes, transfersomes, nanoparticles, and many others which can deliver different natural and synthetic drugs in their structural compartments ,4,5. AboIndeed, the vehicle is important to give texture to the formulation, to permit an easy skin application, and also to drive drug penetration through the skin, reaching deeper skin layers if necessary. It is known that the drug can be included in different types of conventional semi-solid formulation, such as pastes, creams, gels, oils, and ointments; however, these examples of conventional dosage form are often not sufficient to improve the drug’s ability to cross the stratum corneum. On the other hand, the application of liquid suspension of nanocarriers could exceed the skin permeation limit but it can be difficult to apply for the patients, and the formulation could drain away leading to the loss of drug and efficacy.Resveratrol  is a natural phenolic compound belonging to the stilbenoid group, obtained from grapes, berries, peanuts, and red wine ,7. It gaLike many other natural compounds, RSV has not suitable physico-chemical features that guarantee its passage through the stratum corneum after its skin application. In particular, excessively poor solubility in water severely hampers its permeation ability due to the complex skin barrier structure . ConsideLiquid crystal nanosystems have already been proposed as drug delivery systems for the administration of drugs that do not have suitable features for their use in free-form application ,17. In tThe evaluation of mean size, polydispersity index and zeta potential is crucial for confirming the applicability of nanosystems for all administration routes, and above all, for topical application. The data in ® Expert analysis. In This hypothesized stability was confirmed by a Turbiscan LabObserving In The TSI profiles of RSV-loaded LCNs prepared using 0.24 and 0.48 mg/mL are superimposable on RSV (0.72 mg/mL)-loaded LCNs, for this reason we chose to show the stability trend of LCNs prepared with the highest RSV concentrations.Before evaluating the antioxidant efficacy of RSV-loaded LCNs on a human cell model, an in vitro evaluation of safety profile is necessary. This was investigated in human keratinocyte NCTC 2544 cells selected as an in vitro model. The possible cytotoxicity was evaluated as a function of incubation time and the amount of LCNs necessary to reach the effective and chosen RSV concentrations . As can be seen in Considering these in vitro results, LCN-B formulation was excluded from subsequent studies.Oxidative stress, caused by different potential agents such as hydrogen peroxide or UV radiation, is the basis of skin cellular injuries which lead to various pathological conditions such as skin-aging and cancer . Compone2O2 (700 µM) for 1.5 h to induce the oxidant process and damage to cell membranes ) and DSPE-PEG-750 -750]) were purchased from Sigma–Aldrich . Poloxamer P407 , was purchased from BASF . Ethanol was obtained from Carlo Erba SpA , while cellulose membrane Spectra/Por MWCO 10 kDA was obtained from Spectrum Laboratories Inc. .For the in vitro studies, NCTC2544 cells were provided by the Instituto Zooprofilattico of Modena and Reggio Emilia ; all reagents necessary for in vitro studies, such as trypsin/EDTA, culture medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO . Finally, double distilled water was used to prepare all the samples.® glass vial and dissolved in chloroform/ethanol mixture (5 mL—10:90 v/v). Poloxamer P407 was dissolved in water (10 mL) in a second glass vial. To facilitate the solubilization of poloxamer P407, the aqueous phase was maintained for 1 h at 4 °C using magnetic stirring. The organic phase was added dropwise to the aqueous phase while continuously stirring  using the Ultraturrax T25 homogenizer . Once the organic phase has been added, ten homogenization cycles (thirty-seconds each) at 15,000 rpm with 1 min rest between them were carried out. Finally, the formulation was maintained under continuous stirring  at room temperature until any trace of the organic solvent has been removed. For the preparation of RSV-loaded LCNs, the amount of drug  was dissolved in organic phase during the preparation of LCNs.Three batches for each formulation  were prew/w) and CMC 5% (w/w) with the right amount of distilled water [w/w) for the subsequent studies [Empty gels were prepared via simply mixing gelling agents P407 20% (ed water . In the  studies .Dynamic light scattering (DLS) was used to determine mean size, size distribution and zeta potential . For this purpose, a Zetasizer Nano ZS , equipped with a laser diode having a rated output of 4.5 mV at a wavelength of 670 nm and a backscattering angle of 173 °C, was used. For the analysis, each sample was diluted with a suitable medium to avoid multi-scattering phenomena. The same tool was also used for the evaluation of the Z-potential of the vesicular suspensions by applying a Smoluchowsky constant F (Ka) of 1.5 as a function of the electrophoretic mobility of the samples. The various measurements were performed on three different batches (10 determinations for each batch). Results were expressed as the mean of three different experiments ± standard deviation.® Expert was used by evaluating the variations in the delta-backscattering (ΔBS) and the delta-transmittance (ΔT) profiles of empty and RSV-loaded LCNs. The analysis was carried out at room temperature (25 °C) for 1 h. The kinetic stability profiles of the various samples were investigated and compared to each other by expressing the data as Turbiscan Stability Index (TSI) versus time  [For stability analysis, the Turbiscan Labulation) ,47.® Ultra centrifugal filters (4000 rpm for 60 min). The LCNs entrapped within filter was recovered, destroyed by using ethanol, and the entrapped amount of drug was spectrophotometrically determined  at λ max 306 nm [De is the amount of drug detected within centrifuged LCNs, and Dt is the amount of drug used for the LCNs preparation.The entrapment efficiency of resveratrol was evaluated by means of suitable spectrophotometric analysis. The purified RSV-loaded LCNs were centrifuged using Amiconx 306 nm ,49,50. N® Ultra centrifugal filters, and by using the following Equation:Dt is the amount of drug used for the LCNs preparation, and Du is the amount of untrapped drug detected within eluted liquid.To be sure of the obtained results, a counter-test was carried out, analyzing the quantity of drug present in the liquid eluted during centrifugation of LCNs using AmiconExperiments were carried out in triplicate and the results were the average of three different experiments ± standard deviations.In addition, the loading capacity (LC%) was calculated as the percentage of the ratio between the amount of total entrapped RSV and the total weight of the nanostructures.v/v). When cellular confluence reached 80%, the trypsin was used to detached cells that were centrifuged and resuspended. For cytotoxicity evaluation, NCTC 2544 were placed in 96-well culture dishes at a density of 5000 cells/0.2 mL, while for antioxidant studies the cells were placed at a density of 8000 cells/0.2 mL.The human keratocytes NCTC 2544 cell line were incubated using suitable culture medium: DMEM medium added with penicillin (100 UI/mL), streptomycin (100 µg/mL), amphotericin B (250 µg/mL) and fetal bovine serum , calculated as previously reported :(3)Cell Abs T and Abs C the absorbance of treated and untreated (control) cells, respectively. For the quantification, the Varioskan™ LUX microplate reader  was used at λ = 540 nm and λ = 690 nm.Considering 2O2 (700 µM) for 1.5 h to induce oxidative stress. The protective effects of treatments were analyzing by using MTT test as described above and via lactic hydrogenase (LDH) assay. This last test is based on the LDH release obtained when an alteration or disruption of cell membrane occur, as in the case of H2O2 treatment. Pierce LDH cytotoxicity assay kit was used, and its specific protocol was followed. At the end of all treatments, LDH release was analyzed using a spectrophotometer in the cultured medium at λ = 680 nm and λ = 490 nm, and calculated as reported:For the evaluation of protective effects of RSV-loaded LCNs, NCTC 2544 cells were previously treated for 24 h with 0.5 and 1 µM of RSV as free and entrapped form. After this prefixed time, cells were exposed to H2O2 and with or without RSV) (LDH compounds), of LDH spontaneously released from untreated cells (LDH spontaneous) and of LDH recorded after lysis of cells (LDH maximum). The results were reported as the average of five different experiments ± standard deviation.Equation (4) considers the absorbance of LDH released from treated cells  . The analysis is based on the measurement of the particles’ Brownian motion which depends on the viscoelastic feature of our sample. For a better detection of microrheological behavior, each sample (20 mL) was added using latex beads . During the time of analysis (1 h), the backscattered light intensity was detected, obtaining mean square displacement (MSD) as quantification of the sample particle movement as a function of time. Starting from MSD data, the RheoSoft Master 1.4.0.0. was able to provide other microrheological parameters, namely elasticity index (EI) and solid liquid balance (SLB).Dynamic rheological characterization of all gelled samples was carried out by using a Kinexus Pro+ rotational rheometer , equipped with cone-plate geometries . All runs were carried out at 25.00 ± 0.01 °C. A fixed gap between the geometries was pre-set to 1 mm and the excess sample was removed. The obtained rheological data was processed by the rSpace software v.2.1. Compressed air flow (2 bar), pre-filtered through fine and superfine Clearpoint filters , allowed for reaching the pressure able to perform the analysis. Before each analysis, the samples were carefully and gently loaded onto the lower measuring plate of the rheometer and the upper measuring geometry was lowered at very slow speed to prevent the alteration of sample structure. Preliminary tests were needed to define suitable experimental conditions for rheological measurements maintaining within the linear viscoelasticity region (LVR). For this purpose, sweep strain tests (0.01 to 100%) were carried out at a frequency equal to 1 Hz.−1 and ending to 100 s−1. Other analyses were carried out to characterize all gelled samples by means oscillatory test with a frequency sweep ranging from 0.1 Hz to 10 Hz and at controlled stress (1 Pa), obtaining important information about storage modulus G′ (elastic component) and loss modulus G″ (viscous component).Viscosity studies were performed by means of a shear rate ramp starting from 0.1 s2 and 4.75 mL, respectively. To mimic the interaction of nanosystems and human skin, the studies were carried out interposing stratum corneum and viable epidermis (SCE) membrane between donor and receptor compartments of Franz cells. The tissue was obtained from human skin following abdominal reduction surgery, and it was isolated as previously described [The in vitro percutaneous permeation studies were carried out by using dynamic diffusion Franz cells and following a previous consolidated method. The diffusional surface area and the receptor volume of diffusion cell are equal to 0.75 cmescribed . Before escribed .2O:EtOH 70:30 v/v) to guarantee sink conditions, and the medium was constantly stirred and warmed to 37 ± 1 °C. In donor receptors, 200 µL of each formulation were loaded then the donor compartment was sealed by using Parafilm® to induce occlusive conditions. The percutaneous permeation was monitored for 8 h, and at prefixed times, 1 mL of receptor fluid was withdrawn and analyzed using a UV-Vis spectrophotometer. Each aliquot of receptor fluid was replaced with fresh fluid. Three different experiments were carried out and the results were expressed as mean values ± standard deviation.Considering the lipophilicity of RSV, the receptor chamber was filled with a hydro-alcoholic solution  [The skin tolerability and the consequent skin safety profile of proposed gelled formulations were evaluated on healthy human volunteers by monitoring the variation of transepidermal water loss (TEWL) using the C + K Multi Probe Adapter equipped with TewameterGermany) . This apBefore starting the in vivo evaluation, the volunteers were accommodated for 1 h in a day surgery room at 24 ± 1 °C and 40–50% r.h.2) was collected by Tewameter® TM300 and processed by Courage & Khazaka software MPA.Six sites  were marked on the ventral surface of each subject’s forearm, at least 2 cm of distance between each site. Occlusive commercial patches  were used to allow the application of 150 μL of each sample. At fixed times , the patches were gently removed from the skin and data .t-test was used to validate the results, defining * p ≤ 0.05 and ** p ≤ 0.001 as statistically significant. All values are reported as average ± standard deviation.One-way ANOVA was used for statistical analysis of obtained data. A Bonferroni