The solvents used for electrospinning of PVP/PVA nanofibers were ethanol and 90% acetic acid, optimized with central composite design via Design Expert software. NF-2 and NF-35 were selected as optimised nanofiber formulation in acetic acid and ethanol, and their characterization showed diameter of 150–400 nm, tensile strength of 18.3 and 13.1 MPa, respectively. XRD data revealed the amorphous nature, and exhibited hydrophilicity (contact angles: 67.89° and 58.31° for NF-2 and NF-35). Swelling and in-vitro degradability studies displayed extended water retention as well as delayed degradation. FTIR analysis confirmed solvent-independent interactions. Additionally, hemolysis and in-vitro cytotoxicity studies revealed the non-toxic nature of fabricated scaffolds on RBCs and L929 fibroblast cells. Subcutaneous rat implantation assessed tissue response, month-long biodegradation, and biocompatibility through histological analysis of surrounding tissue. Due to its excellent biocompatibility, this porous PVP/PVA nanofiber has great potential for biomedical applications.Electrospinning is a versatile method for fabrication of précised nanofibrous materials for various biomedical application including tissue engineering and drug delivery. This research is aimed to fabricate the PVP/PVA nanofiber scaffold by novel electrospinning technique and to investigate the impact of process parameters  and polymer concentration/solvent combinations influence on properties of electrospun nanofibers. The   In tissue engineering and regenerative medicine (TERM) research, selecting and optimizing a biomaterial that physiochemically replicates the extracellular matrix (ECM) is important . To mimiElectrospun nanofibers play a key role in tissue regeneration owing to their multiple functionalities, such as high surface area, mechanical stability (stiffness and tensile strength), nanoscale architecture, interfibrous porous microstructure, sustained drug delivery and high scale-up potential. There is growing evidence that nanofibrous scaffolds are suitable for TERM because they can recapitulate ECM components, modulate cellular responses, and promote mineralization and osseointegration . ElectroThe dimensions and morphological configuration of nanofibers produced through electrospinning are influenced by a range of factors. These factors can be categorized into three groups: attributes of the polymer solution , operational parameters , and environmental conditions . These factors collectively impact the capability to spin and the morphology of electrospun nanofibers . Hence, Polyvinylpyrrolidone (PVP) stands as a significant amorphous polymer, characterized by its notable biocompatibility, high tensile strength, exceptional solubility in various organic solvents, minimal chemical toxicity, proficient spinnability, and non-hazardous nature. The process of electrospinning has found extensive application in transforming diverse materials into fibers, leveraging PVP’s spinnability and fiber extraction capabilities. Another synthetic polymer that exhibits a commendable aptitude for forming membranes, along with low toxicity in physiological environments and excellent biocompatibility, is polyvinyl alcohol (PVA) . HoweverResponse Surface Methodology (RSM) emerges as a significant approach in modeling the electrospinning procedure due to its capacity to account for the interplay among diverse parameters. RSM is an amalgamation of mathematical and statistical techniques employed in the experimental modeling and analysis of diverse input data that can impact specific outcomes or the quality aspects of a process. Notably, Gu et al., in their research, explored the quantitative correlation between electrospinning process variables and the distribution of average fiber sizes for gelatin and PVA nanofibers, utilizing RSM . In a sein vitro with in vivo degradation was also carried out. There are different sources of lipases in the human body such as leukocytes, present in the wound healing process, with the lipase concentration of healthy adults in the range of 30–190 U/L. Thus, the degradation of the PVP/PVA nanofibers were monitored both in phosphate buffered saline (PBS) and in-vivo subcutaneous rat implantation. The in-vitro degradation kinetics were evaluated through the quantification of weight loss, swelling degree and thermal behavior whereas in-vivo degradation was assessed by using histopathology. Biocompatibility study was investigated using L929 cell lines and hemolysis study.The primary objective of this study is to comprehensively investigate the impact of distinct solvents and process parameters on the diameter of PVP/PVA nanofibers utilizing the electrospinning technique. The research endeavors to optimize various system parameters including concentration, applied voltage (kV), nozzle-collector distance (cm), and flow rate (ml/h) concerning PVP/PVA nanofibers generated in both acetic acid and ethanol solvents. This optimization was conducted using DOE, version 13. RSM with central composite design (CCD) was used to model and optimize the electrospinning process to minimize the nanofiber diameter. The comparison of PVA  and PVP  were purchased from Macklin Biochemical Co. Ltd. (P.R. China). Cell Counting Kit-8 purchased from KeyGEN BioTECH (China). L929 cell lines were gift sample from professor Huang Zhongbing . All additional chemicals and reagents employed in the study were of analytical grade.DOE software version 13 was used to optimize the polymer concentration as well as their nanofibers producibility. In this study, we applied the RSM technique using a CCD to evaluate the influence of polymers solution concentration on nanofiber producibility in ethanol. Face centered central composite design (FCCD) with four center points was used to investigate the relationship between independent variables and responses. Accordingly, the percentage of polymers (%w/v) was considered as the process parameter in the DOE. Two levels, low (−1) and high (+1), were defined for each polymer with different concentrations. As shown in 1) PVP/PVA in acetic acid solvent.2) PVP/PVA in ethanol solvent.To analyze the effect of process parameters in different electrospinning solvents on the producibility and diameter of nanofibers, the selected batches were used for further studies. In this study, a CCD design was used to prepare and optimize the electrospun nanofibers. The main parameters including voltage (kV), flow rate (ml/hr) and distance (cm) with two levels, low (−1) and high (+1), were evaluated for optimizing the nanofibers. As shown in Polymer solutions were prepared in different solvents by mixing 12% PVP and 8% PVA in 90% acetic acid and distilled water under continuous stirring for 8 h to obtain a uniform solution. A mixture consisting of 12% PVP and 8% PVA solutions was meticulously combined in a 50/50 ratio through rigorous stirring to achieve well-blended compounds suitable for electrospinning. The same procedure was replicated using ethanol. These prepared solutions were introduced into a 10 ml syringe fitted with a stainless-steel needle possessing an inner diameter of 0.4 mm. By connecting a positive electrode to the needle, a high-voltage power source was employed. For the collection of nanofibers, a collector enveloped in aluminum foil was employed, positioned at a distance of 8–12 cm from the spinning electrode. To ensure controlled extrusion, the syringe pump’s flow rate was fine-tuned to 1.5–2.5 ml/h, while the solutions were pumped using a high electric voltage ranging from 18–26 kV.The morphology of the electrospun nanofiber batches was analyzed using a SEM model S4800 from Hitachi, Japan. In this investigation, nanofiber samples were affixed to SEM specimen stubs utilizing double-sided carbon tape. Subsequently, a sputter-coating process with Au-Pd was conducted for a duration of 70 s. The analysis was carried out under varying magnifications at an accelerating voltage. The software employed for assessing the nanofiber scaffold diameter was ImageJ. Measurements of fiber diameters were taken at multiple locations for accuracy .n = 3) were cut into 1 mm width and 20 mm length, placed between the two clasps, and subjected to tensile displacement a crosshead speed of 8 mm/min. The stress-strain curve was utilized to calculate the Young’s modulus, tensile strength, and elongation at the point of fracture for the samples.The mechanical characteristics of the nanofibrous mats were assessed using a Universal Testing Machine (UTM)  with a load cell of 5 cN capacity and the standard followed was American Society for Testing and Materials . The nanofibrous samples  was employed. In this process, the sample was initially positioned on a level surface, following which a water droplet was carefully dispensed onto it using a moving needle. The spherical image of the droplet was captured by a digital camera and projected onto a monitor. Subsequently, the contact angle formed between the droplet and the surface of the nanofibers was measured. To ensure accuracy, at least three measurements were taken at different locations on the film and then averaged for comprehensive data analysis.−1 with a resolution of 4 cm−1  spectrometer was used to determine the constituting functional groups of different nanofibers. The structural changes occurring during electrospinning, blending, coating etc. was scanned by FTIR, using standard KBr crystal at room temperature. The spectra were obtained in transmission mode over a wavenumber ranged from 4,000 to 400 cmf 4 cm−1 .The crystalline arrangement of the nanofiber scaffolds were assessed using an X-ray diffractometer . The samples were positioned on quartz zero background holders and subjected to examination with a commercially available XRD system. For detection, a solid-state Germanium detector cooled with liquid nitrogen was utilized, operating with Cu k-alpha radiation at a current of 45 kV and 40 mA. XRD patterns were gathered within a 2θ range spanning from 5 to 60°, with a scanning rate set at 2° per minute .TGA  was used to employed thermal stability of different nanofibers. In a nitrogen atmosphere, all samples were subjected to heating at a rate of 10°C per minute across a temperature range spanning from 25°C to 800°C.2 were precisely weighed (Wi) and then placed into 5 ml plastic tubes containing 4 ml of PBS solution with a pH of 7.4. These tubes were subsequently positioned within a shaking incubator set at 100 rpm and 37°C. The incubation medium was renewed on a weekly basis. At each predetermined time interval, the samples were dried until a constant weight was achieved (Wf)  . The weiWi and Wf represents the initial weight of the sample and the final weight after degradation, respectively.Where 2 pieces, and their dry weight (Wd) was determined using an electronic weighing balance and recorded. Subsequently, the samples were immersed in a PBS (pH 7.4) at room temperature. At specified time intervals, the samples were retrieved from the solution and positioned on tissue paper to eliminate excess water adhering to the nanofiber surface (w) was then promptly measured. All measurements were executed in triplicate, and the water uptake capacity was computed using the subsequent equation:The water retention capacity of the scaffolds was assessed by calculating the swelling ratio. Initially, the samples were sectioned into 1 × 1 cm surface . The weiw represents the weight of the wet nanofiber scaffold and Wd corresponds to the dried weight. A triplicate analysis was conducted, and the average value was adopted as the percentage of water uptake.Where W2. All tubes, including controls and treated samples, were then incubated at 37°C for a duration of 3 h. Upon the conclusion of this incubation period, all tubes underwent centrifugation at 3,000 rpm for 10 min. After incubation and centrifugation, 200 µL of the supernatant from all the samples was transferred to a 96-well plate, followed by the measurement of haemoglobin absorption at a wavelength of 540 nm  of the samples, negative control and positive control.The assessment of scaffolds’ hemocompatibility involved the use of freshly collected blood samples from healthy rats. Blood samples (5 ml) were gathered into tubes coated with heparin and then subjected to centrifugation at 3,000 rpm, leading to the separation of plasma from red blood cell (RBC) pellets settled at the tube’s bottom. With careful removal of the supernatant, the introduction of PBS into the tube ensued. A subsequent centrifugation step was applied to resuspended RBCs for cell isolation. The subsequently purified blood cells were diluted to achieve a 25 ml volume, thus creating an RBC suspension. Following this, 0.5 ml of the RBC suspension was aliquoted into four 1.5 ml tubes. Among these, two tubes were designated as negative and positive controls, wherein 1 ml of PBS and water was added, respectively. The remaining two tubes were subjected to treatment with nanofibers (NF2 and NF35) sized 1 × 1 cmf 540 nm . The folMicroscopic images of the RBC condition were also captured.5 cells/well. The cells will be cultured under standard conditions using appropriate DMEM media, supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin, at a temperature of 37°C and under an atmospheric condition of 5% CO2. Prior to conducting the cell experiments, nanofibers with a standard size of 6 cm2/ml will be sterilized using UV light and 70% alcohol.L929 fibroblast cells will be seeded into 96-well culture plates at a predetermined cell density of 1×10To generate extract solutions, sterile nanofibers will be placed in culture medium (DMEM containing 10% FBS and 1% penicillin-streptomycin). These mixtures will be incubated in a constant temperature shaker at 37°C for 24 h. At the conclusion of the extraction period, samples will be retrieved and the resulting extract solutions will be maintained at 37°C during the subsequent cytotoxicity testing. L929 cells will be exposed to the nanofiber extract solution, and cell proliferation on various nanofibers will be quantitatively assessed using Cell Counting Kit-8  assay at specified time points. The OD value will be measured at 450 nm using a spectrophotometric microplate reader. The experiment will be conducted in triplicate to ensure statistical validity. The acquired data will be analysed to ascertain the impact of nanofibers on L929 fibroblast cell viability on days 1, 3, and 5. Cell viability was calculated using the following formula:2. The nanofibers were trimmed to dimensions of 2 × 2 cm2, after which they underwent a sterilization process involving exposure to UV light for 30 min and treatment with 70% alcohol. Following this, nanofiber-coated 12-well plates were utilized to seed 1×104 cells per well, and subsequent incubation took place. On the third and fifth days, the cell culture media was removed, and the cells were rinsed with PBS (7.4), fixed using a 4% w/v paraformaldehyde solution for 30 min, and subjected to a dehydration process using ethanol (ranging from 10% to 100%). To enable microscopic mineralization analysis, the samples were coated with a layer of platinum using sputter coating and studied using SEM.The L929 cells (murine fibroblast) were cultivated in DMEM culture media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were then incubated at 37°C in an environment with 5% CO2 was inserted into this pouch. Following the polymer implantation, the incision was sutured using a non-absorbable surgical black braided silk thread.All surgical procedures involving animals received approval from the Animal Use and Care Committee of Sichuan University. The nanofibers underwent sterilization through exposure to both 70% ethanol and UV light. Wistar rats, each with a weight ranging from 200 to 250 g, were selected for the animal experiments. These rats were divided randomly into three groups: the first group underwent surgery alone, the second group received PVP/PVA in ethanol (NF35), and the third group received PVP/PVA in acetic acid (NF2); each group comprised six rats. Before the surgical procedures, all experimental animals were anesthetized using chloral hydrate. The dorsal region of the animals was shaved and then sterilized using a solution of 70% ethanol. A sterile surgical blade was employed to create an incision on the dorsum of each animal. Subsequently, a subcutaneous pouch was formed, and an implant measuring 1 × 1 cmAt the end of 1 and 3 weeks, the rats were humanely euthanized. The regions where the implants were positioned were carefully collected; this encompassed both the complete PVP/PVA nanofiber and the adjacent tissue. The collected samples were then immersed in a 4% (w/v) paraformaldehyde solution for fixation, allowing them to be preserved overnight. To prepare the samples for further analysis, they underwent a dehydration process by passing through a series of alcohol baths with varying concentrations, followed by treatment with xylene. Subsequently, the samples were embedded in paraffin and sliced into sections that were 5 µm thick. These sections were mounted onto slides, facilitating histological staining and subsequent imaging.post hoc analysis. Statistical significance was indicated by * for a p-value less than 0.05, ** for a p-value less than 0.01, and *** p-value less than 0.001.The data from all experiments were presented in the form of mean values accompanied by standard deviations (S.D.). Analysis of the data was performed using OriginPro 2019b software. One-way ANOVA was utilized to assess the data across all experiments, and this was followed by the application of the Tukey test for During optimization, it was found that the polymer concentration plays a very important role in the formation of nanofibers; otherwise, nanofibers will form with numerous beads having no uniformity. The relationship between solution viscosity and polymer concentration is highly dependent on the concentration of polymer/solvent system. Each set of PVP/PVA was optimized by DOE for their concentration as mentioned earlier , on the As depicted in In this study, the primary objective was to achieve the production of uniform and smooth nanofibers by optimizing the electrospinning process parameters, namely distance, voltage, and flow rate, utilizing both acetic acid and ethanol solvents. To gain a deeper understanding of the process parameters and to establish a quantifiable relationship between the electrospinning process parameters and the resulting fiber diameter, the RSM was employed . By emplThe influence of voltage on fiber diameter remains a subject of debate and is markedly influenced by the specific characteristics of the polymer and solution employed. Elevated voltage levels can yield disparate outcomes in terms of fiber diameter alteration, as depicted in When examining the acetic acid solvent system at a constant nozzle-collector distance, augmenting the applied voltage leads to a decrease in the average diameter of PVP/PVA nanofibers. However, with a fixed applied voltage, elevating the nozzle-collector distance leads to a reduction in the average diameter (AD) of the nanofibers. This discrepancy can be elucidated by considering the relatively higher boiling point of acetic acid (118°C). In contrast, when using an ethanol solvent, decreasing the applied voltage or increasing the nozzle-collector distance diminishes the strength of the electric field, resulting in less acceleration and stretching of the jet. Consequently, this leads to the generation of larger PVP/PVA nanofibers during the electrospinning process. Subsequent to the dispersion and division of an unstable jet, solvents with lower boiling points, such as ethanol (78.37°C), swiftly undergo evaporation .The rate at which the polymer solution flows within a given timeframe constitutes an additional element impacting the quality of nanofibers. As evidenced by the findings and outcomes presented in However, it is essential to note that an excessive flow rate not only augments nanofiber agglomeration but also gives rise to bead formation within the fibre structure due to insufficient time for solvent evaporation.The distance between the nozzle and the collector constitutes an additional parameter influencing the regulation of nanofiber morphology and diameter. Achieving precise control over polymer solution evaporation before the fiber reaches the collector necessitates the optimization of this distance. Drawing insights from the outcomes and findings presented in A noteworthy observation entailed a minimal fibre diameter of 179.224 nm, achieved at a voltage of 26 kV, flow rate of 2.5 ml/h, and distance of 8 cm in the acetic acid solvent. Correspondingly, the average diameter of electrospun PVP/PVA nanofibers, measuring 271.065 nm, was attained using a voltage of 26 kV, flow rate of 2.5 ml/h, and distance of 8 cm in an ethanol solvent. Ethanol exhibited promise as a suitable solvent for PVP/PVA, yielding electrospun fibres with a broad range of diameter distribution conducive to microfiber production. Comprehensive analysis substantiated that polymer concentration, solvent selection, and process parameters all exerted noteworthy impacts on the resulting diameter values.Mechanical properties hold paramount importance for nanofibers, as the underlying matrix must possess robust mechanical strength to effectively facilitate tissue repair. Evaluating the scaffolds’ tensile strength (TS) measured in MPa was accomplished through the assessment of strain-stress curves. The determination of hydrophilicity and wettability of the NF2 and NF35 scaffolds involved employing the static water contact angle method through  instrument. This methodology facilitated the measurement of the contact angle for the optimized nanofiber formulations. Wettability holds pivotal importance as it significantly impacts a scaffold’s mechanical stability and its interactions with adhering cells. In the course of contact angle analysis, the shape of the liquid droplet was contingent upon variables such as liquid surface tension, gravity, and the density difference between the liquid and the nanofibers. The outcomes of the contact angle measurements, visualized in Hydrophilicity is a key determinant of bioactivity, rendering hydrophilic scaffolds preferable for tissue engineering applications. Specifically, a surface is considered hydrophilic if the contact angle measures below 90° and hydrophobic if the angle exceeds 90°. For NF2 and NF35, the contact angles were 67.89° and 58.31°, respectively. This divergence in contact angles between NF2 and NF35 might be attributed to the influence of solvent effects and nanofiber diameter.-1 were recorded in FTIR spectra of PVA/PVP nanofibers. -1 along with strong intensity due to the stretching vibrations of hydroxyl group in both the nanofiber. But the intensity of peak of hydroxyl group in NF35 was more, it may be due to the presence of more hydrogen bonding in the presence of ethanol. The C-H bending at 842 cm-1 in PVA polymer, the band at 1,077 cm-1 confirm the presence of C-O vibration of PVA-PVP. In addition, the presence of a 1,631 cm-1 peak was due to stretching vibrations of the carbonyl group present in PVA. The band at about 1,279 cm-1 corresponds to C-O stretching of acetyl groups present on the PVA backbone. The appearance of C-O stretching is due to the semi-crystalline nature of the blends. A band at 1,373 cm-1 is attributed to C-N bond, mainly from the functional group of PVP. The vibration band at about 1,636 cm-1 corresponds to C-O symmetric bending of PVA and PVP. The band corresponding to CH2 asymmetric stretching vibration appeared around 2,928 cm-1 in PVP.Different absorption bands within 4,000–500 cmAn XRD study was conducted to investigate electrospinning-induced crystalline changes and provide details regarding the occurrence of complex formation between different polymers. The XRD patterns of the PVP/PVA blends with different solvents are shown in The thermal stabilities of the selected NF2 and NF35 nanofibers in different solvents were examined by TGA, as shown in The degree of degradation of NF 2 and NF 35 scaffold was also determined by observing the mass change of the samples after immersion in PBS. The degradation behavior of the scaffold’s during incubation is depicted in The swelling behaviour exhibited by the scaffolds (NF2 and NF35) underscores their capacity to facilitate nutrient and waste exchange between the cellular environment and the cells embedded within the scaffold, a crucial aspect in the creation of artificial tissues. Swelling, in this context, signifies the ability to imbibe moisture and establish stability within biological systems. It offers potential as a carrier material for cell proliferation and differentiation, consequently playing a vital role in tissue engineering. Upon implantation, biomaterials interact with the surrounding fluids, initially by uptaking them, thus promoting the degradation process. The water uptake makes the materials more flexible and promotes changes in the dimensions of the implant material.As portrayed in The hemolysis test is valuable in assessing blood compatibility as it reflects cytotoxicity. RBCs can lyse and release biomolecules like hemoglobin when they encounter water or foreign substances, due to osmotic stress. Damaged RBCs can attract platelets, accelerating coagulation and hindering tissue regeneration . An optiThe impact of PVP/PVA nanofibers, specifically NF2 and NF5, on L929 fibroblast cell viability was assessed through a CCK-8 assay. The cell viability results from day 1 displayed more than 80% cell viability for NF2 and 90% for NF35 . Remarkain vivo biocompatibility and potential therapeutic applications of these nanofibers. This procedure facilitated the exploration of nanofiber-host tissue interactions, cellular responses, and overall biodegradation processes within a controlled condition. The chosen subcutaneous implantation technique, along with precise suturing and controlled animal housing, ensured a more realistic representation of the nanofibers’ interaction with the surrounding tissue. After 3 weeks of subcutaneous implantation, the nanofibers’ macroscopic appearance was similar to that of pre-implant hydrated fibre  value, along with its adjusted counterpart (adjusted R2). In this context, a model is considered valid if its R2 value is equal to or greater than 0.60, as affirmed by the model under consideration. The predictive capability of the model for new observations is reflected in the Predictive R-squared (Pred-R2), while the regular R2 and adjusted R2 values reflect the model’s alignment with empirical outcomes. The Pred-R2 and Adj-R2 values stand at 0.9323 and 0.9817, respectively for nanofiber producibility . This value signifies the proportion of the total variability that the regression model is able to explain. The R2 values obtained in this study confirmed the models’ validity, as they exceeded the threshold of 0.60. Exploration of the comprehensive impact of these parameters on all produced nanofiber batches is elaborated upon in the subsequent discussion.These process parameters were subsequently evaluated using ANOVA, with the summarized outcomes presented in SEM was utilized for the morphological analysis of nanofibers, encompassing characteristics such as fiber shape, diameter, and surface structure. This inspection exposed the vital role of nanofiber topography in early cellular processes, such as adhesion and proliferation . Upon atin-vitro degradation studies indicates that these nanofibers possess the necessary characteristics for tissue regeneration. The in-vivo evaluation, including subcutaneous rat implantation and histological analysis, underscored the exceptional tissue biocompatibility of the PVP/PVA nanofibers over a month-long period. Furthermore, the in-vitro cell lines studies indicated nanofibrous material promoted cell growth/proliferation.The obtained nanofibers exhibited a consistent and controlled diameter range of 150–400 nm, indicative of a precise electrospinning process. Mechanical testing revealed notable tensile strength values ranged from 13.81 to 18.3 MPa for PVP/PVA nanofibers, highlighting their structural integrity and suitability for regenerative medicine and drug delivery applications. XRD analysis underscored the amorphous nature of the PVP/PVA nanofibers, suggesting their potential for enhanced drug loading and release kinetics. The FTIR results provided evidence of consistent and solvent-independent interactions, reinforcing the reliability and reproducibility of the fabrication process. The significant hydrophilicity exhibited by the PVP/PVA nanofibers, as evidenced by contact angle measurements, bodes well for their interactions with biological systems. This property was further corroborated by the extended water retention observed in swelling studies, aligning with the requirements of sustained and controlled drug release applications. Moreover, the 2-week biodegradation timeframe revealed through in-vivo biocompatibility of the PVP/PVA nanofibers was assessed by histological analysis of surrounding tissue, which underline the exceptional tissue biocompatibility and appropriate in-vivo biodegradation. This outcome holds great promise for their application in tissue engineering, where biocompatibility and biodegradability are paramount.PVP/PVA nanofiber scaffold was successfully prepared by using electrospinning method and explored the influence of process parameters such as concentration, voltage, nozzle-to-collector distance, flow rate and solvent selections on the properties of these nanofibers. Through a meticulous investigation using CCD and DOE, the optimal conditions for electrospinning of PVP/PVA nanofibers using ethanol and acetic acid were determined, leading to remarkable insights into their potential application in biomedical field. The physiochemical property of obtained nanofibers exhibited hydrophilic nature with porous structure similar to that found in native ECM. The


In this study, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, and 10 wt% Ca admixtures with copper oxide were investigated. The determined optimal calcium dopant concentration was 4 wt%. XRD analysis was used to reveal the chemical composition of the produced layers. It was found that a calcium dopant does not change the layer composition but improves its electrical parameters. Based on UV-Vis spectra, the band gap energy and Urbach energy were calculated. The morphology of produced thin films was described as smooth and nanocrystalline, corresponding to a grain size calculated based on the Scherrer equation. Finally, it was shown that the developed protocol of low-resistivity copper oxide deposition via the spray coating technique can be successfully implemented in heterojunction solar cell production. The I–V parameters of Ag/n-type CzSi/REF:CuOx and 4Ca:CuOx/Carbon were collected, and the achieved efficiency was 2.38%.In this report, the morphological, optical, electrical, and photovoltaic properties of copper oxide and calcium-doped copper oxide thin films produced via the spray coating method were studied. The thermal post treatment at 300 °C in an inert atmosphere allowed us to obtain a single phase of Cu Nyborg 017 cm−3 . Also, s 330 Ωcm . Another 330 Ωcm . Meanwhi019 cm−3 . Chafi e−3 }. Atomic force microscopy  was used for surface roughness determination. The thickness of the layers was determined via transmission electron microscopy (TEM) using a TECNAI G2 F20 (200 kV) FEG Thermo Fisher Scientific. The thin film preparation was carried out using the focused ion beam FIB technique. The I–V parameters of solar devices were investigated using a Photo Emission Tech AAA class solar simulator under standard test conditions.The manufactured layers were characterized optically using a Perkin Elmer Lambda 950 S UV-Vis spectrophotometer. The resistivity was calculated based on sheet resistance measurements determined using a four-point probe (PIE SPC-90). The qualitative phase analysis was carried out using X’Pert Pro , with a cobalt anode lamp (KαCo λ = 1.7909 Å). The measurements were performed using grazing incidence X-ray diffraction (GIXRD). The obtained diffractograms were analyzed by means of the X’Pert High Score Plus software (v. 3.0e) with a dedicated Inorganic Crystal Structure Database—ICSD . The XPS analyses were carried out in a PHI VersaProbeII Scanning XPS system using monochromatic Al Kα (1486.6 eV) X-rays focused on a 100 µm spot and scanned over an area of 400 µm × 400 µm. The operating pressure in the analytical chamber was less than 3 × 102O by means of the spray coating method is annealing the layer in an oxygen-free, protective atmosphere at 300 °C. It was proven that even at 250 °C in air, the as-deposited copper oxide layer A:CuOx consists of a CuO phase. This results in higher film resistivity of 23.2 Ωcm, a smaller grain size of 6.17 nm, and much a higher Urbach energy of 693 meV compared to REF:CuOx treated at a high temperature in nitrogen. The REF:CuOx is characterized by a resistivity of 21 Ωcm, a larger grain size of 10.42 nm, and a lower Urbach energy of 240 meV, which suggests lower structural disorder. What is more, in order to reduce the resistivity of REF:CuOx, the calcium admixture was implemented at concentrations of of 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, and 10 wt% from calcium acetate. The admixture was introduced directly to the copper acetate precursor solution. The layer resistivity decreased to 12 Ωcm for the 4 wt% calcium admixture. Also, the charge carrier concentration improved to 8.48 × 1016 cm−3. It should be emphasized that the Ca dopant had no effect on the phase composition of the manufactured Cu2O layer. However, it increased the transmittance of the copper oxide thin film and reduced its reflectance. The band gap energy decreased from 2.55 eV for REF:CuOx to 2.45 eV for 4Ca:CuOx. It was found that calcium doping inhibits the growth of oxide grains, which is why the grain size determined by the Scherrer equation is slightly smaller, at 8.82 nm, for the doped CuOx compared to the reference REF:CuOx. The layer thickness indicated by the TEM cross-section is about 40–50 nm for both tested coatings, REF:CuOx and 4Ca:CuOx. It was also confirmed that the developed protocol of low-resistivity, calcium-doped copper (I) oxide production via the spray coating method can be useful for solar cell application. The produced heterostructures based on n-type CzSi and spray-coated copper oxide revealed that calcium doping improved the electrical parameters of solar devices. The maximum efficiency achieved was 2.38% with an Isc of 0.98 mA and a Voc of 275 mV. This work will be continued, and the manufactured copper oxide will also be deposited on n-type oxides such as zinc oxide to produce thin-film heterojunction solar cells. The presented results show that the crucial step in the preparation of Cu


These computational approaches represent the toolbox of computational chemogenomics , IDH1-R132C, an oncogenic metabolic enzyme. The inhibitors were identified by means of docking-based virtual screening of a commercial synthetic library with 1.5 million compounds and cellular inhibition assays. The most promising compound (with the identifier T001-0657) showed high selectivity for cancer cells with the IDH1-R132C mutation and could be further developed as a tool compound to further investigate the biological role of the mutant IDH1-R132C. The authors explored the potential molecular mechanism of the newly identified compound and structural domain of IDH1-R132C with molecular dynamics simulations and free energy calculations. Ghosh et al. discuss the computational characterization of thirty aryl benzoyl hydrazide derivatives with experimental evaluations as inhibitors of the RNA-dependent RNA polymerase enzyme of the H5N1 influenza virus. The authors investigated the structural requirements for antiviral properties of the compounds with 2D-quantitative structure-activity relationship (2D-QSAR), 3D-QSAR, structure-based pharmacophore modeling, molecular docking, and molecular dynamics simulations. Specifically, molecular docking was employed to generate a structure-based pharmacophore mapping. Molecular dynamics was conducted to assess the dynamic stability of the docked ligands at the proposed binding site of the receptor. The binding interactions obtained from the molecular dynamics simulations had a good agreement with the findings of the 2D-QSAR, 3D-QSAR, and pharmacophore modeling. Cofas-Vargas et al. explore the binding of the fungal antibiotic aurovertin, which is an exogenous allosteric inhibitor of the enzyme FOF1-ATP synthase. This enzyme carries out several major regulatory functions in the cell membrane so that its malfunction has been associated with a growing number of human diseases. It is also a promising drug target to combat antibiotic resistance. To characterize the protein-ligand interactions, the authors employed mixed-solvent molecular dynamics, end-point binding free energy calculations, and free energy calculations. The findings of this work helped to provide insights for the structure-based design of novel allosteric drugs targeting FOF1-ATP synthase sites of exogenous inhibitors. Yang et al. identify dodoviscin A as an inhibitor of the extracellular signal-regulated protein kinase 2 (ERK2). Toward this goal, authors conducted docking-based virtual screening of a natural product collection prefiltered with estimated ADMETox properties. As part of the structure-based virtual screening, the authors compared the calculated binding energies of the screening collection with desirable pharmacokinetic characteristics with magnolipin, a known inhibitor of ERK2. The putative binding mode and stability of dodoviscin A with the binding site of ERK2 were explored with molecular dynamics simulations.Liu et al. introduce the free web-based database of pharmaco-omics for cancer precision medicine (DBPOM). The database provides the reversed and adverse effects of 19,406 small-molecules and drugs and 509 drug combinations on the patients of five cancer types based on the genomics and transcriptomics information of 28 kinds of cell lines and 3078 cancer samples. In their work, the authors also describe a methodology to predict the drug’s efficacy to reverse or enhance their cancer-associated gene expression change. Liu et al. anticipate that the webserver DBPOM will become a valuable platform for drug development, drug mechanism studies and the discovery of new therapies.in silico approaches to pharmacological research and drug discovery. It is expected that the article collection will motivate, inform, and provide direction to the researchers and students in the field.In conclusion, the authors, the Research Topic Editors, and the journals of Frontiers in Pharmacology and Frontiers in Drug Discovery anticipate that this Research Topic contributes to illustrate the progress made in the development, validation, and successful applications of


Enhancing the performance of adhesively joined compositecomponentsis crucial for various industrial applications. In this study, polyamide66 (PA66) nanofibers produced by electrospinning were coated on unidirectionalcarbon/epoxy prepregs to increase the bond strength of the composites.Carbon/epoxy prepregs with/without PA66 nanofiber coating on the bondingregion were fabricated using the autoclave, which is often used inthe aerospace industry. The single lap shear Charpy impact energyand Mode-I fracture toughness tests were employed to examine the effectsof PA66 nanofibers on the mechanical properties of the joint region.Scanning electron microscopy (SEM) was used to investigate the nanofibermorphology and fracture modes. The thermal characteristics of Polyamide66 nanofibers were explored by using differential scanning calorimetry(DSC). We observed that the electrospun PA66 nanofiber coating onthe prepreg surfaces substantially improves the joint strength. Resultsrevealed that the single lap shear and Charpy impact strength valuesof the composite joint are increased by about 79 and 24%, respectively,by coating PA66 nanofibers onto the joining region. The results alsoshowed that by coating PA66 nanofibers, the Mode-I fracture toughnessvalue was improved by about 107% while the glass transition temperatureremained constant. The performance ofjoints is influenced by various factors, including environmental conditions.Such as type of adhesive, surface treatment, temperature, load, andhumidity.8 Traditionally, two established methods exist to join components,i.e., mechanical fastening and adhesive bonding. Mechanical connections,achieved through bolts or rivets that involve drilling, can lead toproblems in composite materials. When composite materials are drilled,delamination, fiber structure degradation, and stress concentrationscan occur around the holes. To address these concerns, adhesivelybonded joints have gained popularity due to their inherent advantagesover mechanical fastening. The use of adhesively bonded joints resultsin a more uniform load transfer across a larger area, eliminatingthe need for fastening holes and fasteners. This, in turn, reducesstress concentrations and weight gain in the overall structure. Moreover,in these applications, the adhesion properties of the adhesive arestrengthened by applying surface treatments to the bonded material.In the literature, the strength of the joining processes is evaluatedby the lap shear test. Researchers have identified three primary methodsof adhesive joining for manufacturing composite structures.9 The first method involves a simultaneous joiningand curing process of both parts, known as cocuring. This can be carriedout with or without the use of an adhesive, as both parts are uncuredduring the process. The second method is cobonding, where a curedpart is attached to an uncured part using an adhesive on the joiningsurface. The third method is secondary bonding, where two cured sameor different parts are joined by applying an adhesive to the joiningsurfaces. According to the majority of researchers, the strongestbond strength performance is typically attained when employing cocuringor secondary bonding methods.8 Particularlywhen joining complex structures, secondary bonding has demonstratedsuperior performance compared to the other two methods, as indicatedin previous studies.11 In-depth investigations by researchers have focusedon understanding failure mechanisms occurring at the joint interfaces.Adhesive failure, caused by the breakdown of the bond between theadhesive and the structure, is one of the common problems encountered.12 Interestingly, studies have indicated that increasing the adhesivethickness contributes to better adhesion of the adhesive to the surface.13Composite materials arematerials with higher strength-to-weightratio characteristics. For this reason, the use of composites continuesto increase, especially in the aviation industry.19 Researchers have actively explored the use of nanofibers as an interfacecomponent during the fabrication of composite materials from prepregs,with the aim of enhancing material strength. Electrospinning is themost effective method of producing nanofibers from polymer materials.Nanofiber diameters can be precisely tuned by changing the parametersused in this method . This level of control over the electrospinning process contributesto the versatility and tailorability of nanofibers for various compositeapplications.21 Extensive research has been conductedon electrospun nanofibers due to their remarkable reinforcing capabilitiesin polymer composites. This is primarily attributed to their ultrafinesize, exhibiting high mechanical strength, and possessing an exceptionallylarge surface area. The unique combination of these properties makeselectrospun nanofibers promising candidates for enhancing the performanceof polymer composites in various applications. Nanofibers producedby electrospinning can be prepared from various polymer solutionsand by incorporation of various fillers. It was shown by multiplestudies that the addition of nanofibers to composite structures enhancesthe mechanical properties.22 Recent studieshave introduced the utilization of thermoplastic nanofibers as a meansto enhance composite strength while simultaneously preserving thein-plane mechanical properties. There are many studies dedicated toexploring the mechanical performance of different materials. For example,adding poly e-caprolactone nanofibers produced by electro-spinningwas found to increase the fracture toughness of composite materials.23 Similarly, adding polyacrylonitrile (PAN) nanofibersbetween carbon fabrics was shown to increase the load capacity ofpin-bonded composite laminates.24 Bilgeet al. showed that incorporating P(St-co-GMA) nanofibersinto composite structures yields an 18% increase in the tensile strength.25 Moreover, the research demonstrated that byadding nanofiber interlayers, comprising 9 wt % of the composite,to regions exposed to high stress, the maximum breaking stress wasfurther improved. These findings highlight the potential of nanofiberreinforcement in increasing the mechanical performances of compositesin critical areas subjected to significant stress. Furthermore, theaddition of nylon-6 (N6) to composite materials demonstrated remarkableenhancements in Young’s modulus.26 Specifically, the N6/YD composite exhibited a 20.5% improvement,the N6/YDJR composite showed a 49% enhancement, and the N6/JR compositedisplayed an extraordinary 1700% increase in Young’s modulus.Additionally, the study highlighted that incorporating N6 nanofiberscritically improved the thermal stability of the epoxy resin matrices.This indicates the potential of nanofiber reinforcement in enhancingboth mechanical performances and thermal performance of epoxy-basedcomposites. Saz-orozco et al. conducted an investigation into theMode I fracture toughness of glass fiber/vinyl ester (GF/VE) compositesand explored the effects of interleaving with polyethylene terephthalate(PET) and polyamide (PA).27 PA nanofibersshowed a better improvement compared to PET nanofibers. The propagationvalue of the PA-reinforced composite increased up to 90% and the initialfracture toughness value increased by 59%. In recent times, the focusof research has shifted toward electrospinning PA66 nanofibers becauseof their exceptional characteristics, which surpass those of othermaterials used in similar applications. Mechanically strong PA66 nanofibershave gained attention for their impressive attributes, including excellentmanufacturability, remarkable fiber-forming capability, melting inhigh temperatures, and compatibility with resin. These superior propertiesmake PA66 nanofibers an attractive choice for various applicationsin different forms of polyamides, leading to increased interest andexploration in the field of nanofiber reinforcement.Recently, there has been increasing interest in the use ofan electrospinningmethod to improve the mechanical and chemical properties of materials.28 Beckermann and Pickering studied the effectson interlayer fracture toughness, Mode I and Mode of autoclaved unidirectional(UD) carbon/epoxy composite specimens by adding polymer nanofibersinterspersed in the interlayers.29 Aljarrahand Abdelal investigated an increase of up to 25% in the interlayerMode I fracture toughness of carbon/epoxy laminates using differentnanofiber configurations.30 In addition,different studies have been conducted in the literature to investigatethe surface-wetting behavior of nanofibers. The polyamide-66 nanofiberswere obtained to exhibit high absorbency.31 Studies reported that the PA66 nanofiber coating resulted in enhancedwetting, leading to a substantial decrease in the contact angle. Theseresearch findings demonstrate the potential of PA66 nanofibers inenhancing material properties and fracture toughness in various compositeapplications.33 Ahmadloo et al. conducted a studyin which they included different PA66 solution concentrations  to increase the Mode I fracture toughness of nanocomposites.34 The research revealed significant improvementsin the maximum failure load of the material as the nanofiber coverin the epoxy increased. This suggests that the addition of PA66 nanofibercovers positively influences the fracture toughness characteristicsof the epoxy-based nanocomposite. In another study by Nan Zheng etal., PA66/PCL nanofibers were used as an interlayer to increase thefracture toughness of carbon fiber/epoxy composites35 Saeedifar et al. observed that PA66 nanofibers reduce thetoughening ability of C/E composites at high temperatures.36 Overall, researchers presented a wide rangeof advantages of electrospun PA66 nanofibers, but their use for secondarybonding is very limited. Depending on production conditions and partdimensions, secondary bonding becomes the technically optimal methodin many cases. However, secondary bonding frequently suffers fromadhesive and substrate bonding failure. While increasing the adhesivethickness was found to enhance the adhering of adhesive to the surface,the resulting weight gain is undesired. For such a case, secondarybonding is a very promising solution to enhance interfacial bonding.A largenumber of authors explored different uses of differentPA66 solutions and reported observed advantages. In their research,Sanatgar et al. explored the electrospinning of PA66 using differentsolution ratios. The presence of chloroform (18% by weight) in thePA66/formic acid solution caused a reduction in solution crystals.37 the present study makes a unique contribution to a better mechanicalperformance understanding of electrospun PA66 nanofibers incorporationinto the joint region of secondary bonded carbon fiber reinforcedpolymer (CFRP) composite parts. Unidirectional (UD) carbon/epoxy prepregfabrics employed commonly in aerospace applications were used withand without PA66 nonwoven coatings to fabricate composite laminates.The nanofibers produced were directly coated onto carbon fabric. Inthis study, the morphology of the nanofiber layer formed by electrospinningPA66 was investigated using SEM to assess its homogeneity and absenceof bead-like structures. The thermal properties of the PA66 nanofiberlayer were measured using DSC. For the experiment, reference and PA66-coatedcomposite samples were produced by using the autoclave technique.The mechanical strength of the joints was determined by various tests,including the single lap shear test, Charpy impact test, and Mode-I(DCB). Additionally, the joining region failure modes were investigatedto understand how the PA66 nanofiber coating affects the joining performance.The shear strength limits of the joint were analyzed in order to measurethe impact of electrospun PA66 nanofibers on the performance of thejunction. These comprehensive evaluations were conducted to assessthe potential enhancement in mechanical properties and toughness ofthe composite joints by the inclusion of electrospun PA66 nanofibers.Impact resistance and interfacial strength are observed to be improvedby CRFP, which is strengthened by the PA66 nanofibers.As an extension of our previous work,22.12, were employed inthe study. As the adhesive, the FM300 K film adhesive was utilized.In the electrospinning process, PA66 pellets sourced from Sigma-Aldrich-429171were used. For dissolving the PA66 pellets, formic acid (Sigma-Aldrich-27001)and chloroform (Sigma-Aldrich-24216) were selected as solvents, followingthe practices described in the literature.28UD carbon fiber/epoxy prepregfabrics, whose unit weight is 350 g/mAfter the curing process, 0.142 mm was read as the ply thicknessof the UD prepreg, while 0.16 mm was obtained as the average thicknessof the FM300 K adhesive, as detailed in 2.215 The electrospinningdevice setup depicted in Prior to preparing the solutions, PA66 pellets were subjected toa moisture removal process by heating and keeping them at 80 °Cfor 24 h. At room temperature, the solution ratio was establishedby dissolving a 10% weight ratio of PA66 pellets in 100 mL of formicacid/chloroform (75:25 v/v). The inclusion of chloroform in the PA66/formic-acidsolution creates an increase in solution viscosity, promoting theproduction of more uniform nanofibers. The selection of this specificconcentration was based on the outcomes reported in a previous studyconducted by the researchers.28 The flow rate of the PA66 solution was set at 18 mL/h, with eachnozzle operating at 1.0 mL/h. For the electrospinning process, theresearchers optimized the applied voltage to be 30 kV while maintaininga nozzle-to-fiber distance of 12 cm. Thermal properties of the electrospunPA66 veils were analyzed by Differential Scanning Calorimetry (DSC).Under a nitrogen atmosphere, a spun sample extracted from the surfaceof the carbon underwent heating between room temperature and 350 °C,where a heating rate of 108 °C/min was performed. For the evaluationof wetting angles, by utilizing the device of the KSV Attension Theta,measured contact angles were conducted in the laboratory. Wettingangles have been obtained at three distinct locations on each surface.Two 50 mL syringes, which were filled with thePA66 polymer liquid,were connected to the propellant pump. To achieve the production ofuniform and bead-free PA66 nanofibers, the researchers identifiedthe optimal parameters through a combination of their experience andinsights gleaned from relevant recommendations in the literature.2.3s order with and without electrospun PA66 nanofiber coatings(We employed the autoclave technique to manufacture composite laminatesof UD prepreg (HEXPLY - M91/IM7/34RC/UD/194/12K) CFRP at [45/–45/45/90/–45/0]coatings2. The faPA66 nanofibers (coated for 10 min and 0.021 mmthick after coating)were added only to the first layer (joining zone) of the 12-ply prepregs2b. The r2 and an average thickness of4.8 mm (12 layers of UD prepreg +3 layers of Film adhesive +12 layersof UD prepreg) were trimmed according to the test specimen dimensionsdescribed within ASTM and ISO standards.The high-viscosity FM300 K film adhesive is usedto bond the UDparts together. The composite laminates with PA66 incorporated intothe joint area were bonded together as described in The single lap sheartest specimens are illustrated in 2.43839 Charpy impact strengthis determined by the ratio of the energy absorbed during the impacttest to the notched area of the sample.We performed Single-lapshear, Charpy impact energy, and Mode-I tests on adhesively joinedunidirectional Carbon fiber composites. GIC) value was calculated using the ASTM standardand the Modified Beam Theory data reduction method.41The Mode-I laminar fracture toughness of the compositespecimenswas evaluated by using the DCB test. The tests were performed on aShimadzu AGS-X instrument using the ASTM D5528 test standard. Theconfiguration of the DCB test specimens is illustrated in 3Based on theprocedure described, we obtain a uniform coating ofelectrospun nanofibers onto the surface of carbon prepregs. Afterelectrospinning, nanofiber deposition was observed as a color changeof the prepreg surfaces 1b. The rTm) and the glass transition temperature (Tg), respectively.The DSC curves of the PA66 nanofibers are givenin We performed lap shear tests according to ASTMD5868 as the load–displacementcurves are given in When the fracture surface images and the test resultswere examined,it was proven that adding PA66 nanofibers to the joint region causeda strong improvement in the strength of the joint region. This structure,prepared by adding PA66, was identified as an excellent method toincrease strength by reducing the fragility of the joining zone.2, respectively (The Charpy impact energies of the reference and PA66-added sampleswere 111.2 and 137.8 kJ/mectively 13. When ectively 14. The fBased on the Mode I test results, Fmax data forthe reference compositespecimens and the PA66 added composite specimens were determined as64.5 and 79.1 N, respectively. During the testing process, PA66 nanofibersplayed a vital role in resisting crack propagation, consequently increasingthe Mode I value of the composite material 15. The nGIC and delaminationlength curvesof PA66 unmodified and PA66 modified test specimens are shown in GIC value of the PA66 unmodified test specimenswas calculated as 0.1756 kJ/m2. The average GIC value of PA66-added composites was calculated as 0.3640kJ/m2. When the samples with and without PA66 were compared,it was observed that the Mod-I value was improved by approximately107%. The GIC and delamination lengthcurves for both reference and PA66 added composite specimens are presentedin GIC value of the nonadded compositeswas calculated to be 0.1756 kJ/m2. In contrast, the mean GIC value of the PA66-added composites was determinedto be 0.3640 kJ/m2. By comparing the PA66-added compositesamples with the nonadded composite samples, it becomes evident thatthe Mode-I value was significantly improved by approximately 107%.This substantial enhancement in fracture toughness demonstrates thepositive impact of incorporating PA66 nanofibers into the compositematerial. The PA66 nanofibers effectively contributed to strengtheningthe interlaminar bonding and increasing the resistance to crack propagation,leading to a significant improvement in the composite’s abilityto withstand Mode-I loading conditions. The result indicates thatthe addition of PA66 nanofibers has notably enhanced the overall mechanicalperformance and fracture resistance of the composite material.The The fracture surfaces of the nonadded referenceand PA66 addedsamples after the double cantilever beam testing can be seen in 4In order to developthe mechanical capabilities of CF/EP composites,PA66 nanofibers were incorporated into the bonding region of compositejoints as a content of this study. To determine the impact of PA66interleaving systems on the mechanical performance of CF/EP composites,some mechanical test studies were performed. It can be read from resultsthat strength in the Single lap shear test and Charpy impact testare improved by approximately 79% and 24%, respectively, by incorporationof the PA66 nanofibers into the joint region. The results also showedthat by using PA66 nanofibers, the Mode-I fracture toughness valuewas increased by approximately 107%. By combining PA66 nanofiberswith the joint regions of the composites, some properties of the compositeswere enhanced, such as impact damage energy absorption, shear strength,and fracture toughness.


Antimicrobial resistance (AMR) is currently regarded by the World Health Organization (WHO) as one of the most significant risks to global public health. The most critical causes of AMR infections in humans are the misuse and overuse of antimicrobials in humans and farmed animals. The rising global demand for food of animal origin encourages the increase of animal production worldwide, especially in developing countries. Simultaneously, current farming practices often extensively use antimicrobials on animals, influencing bacterial AMR incidence. This study aims to evaluate the correlation between antimicrobial use (AMU) in farmed animals and the detection of AMR infections in humans, the effects of enforcing laws in animal farming in a country on AMR situation in the neighbors, and the potential of AMR to spread from one country to another. Using data from 30 largest animal-producing countries in different regions of the world, between 2010 and 2020, and a Spatial Durbin Model (SDM), we found that AMU in farmed animals increases AMR in humans and there is a spatial dependence between countries regarding AMR spreading. Such findings indicate that a globally coordinated strategy regulating AMU on farmed animals may reduce AMR emergence and worldwide spreading. •Antimicrobial use (AMU) in farmed animals is positively correlated with antimicrobial-resistance (AMR) in humans.•There is a spatial dependence between countries regarding the spread of AMR infections in humans.•A globally coordinated strategy regulating AMU in farmed animals contributes to AMR reduction at a global scale. AntimicThe growing demand for food of animal origin  has contModern animal farming faces the dual challenge of increasing production and combating antimicrobial resistance (AMR) due to the ongoing global demand for food of animal origin. . While cThe literature agrees that the inappropriate AMU in farmed animals contributes to AMR in humans. Still, the details and size of this connection and how it is influenced by other factors, such as current AMR levels and social, economic, and epidemiological factors, need to be unraveled . Most stTherefore, correctly understanding the connection between AMU in farmed animals and AMR in humans is crucial for acting and safeguarding antibiotic efficacy  and reduEscherichia coli (E. coli) and Staphylococcus aureus (S. aureus) [1.How does AMU on farmed animals in a country affect AMR in humans in that country?2.How do policies regarding AMU on farmed animals in one country influence AMR in humans in neighboring countries?3.How does AMR in humans cross over national borders?We selected nine animal species that are the primary sources of animal food for humans worldwide: cattle, chickens, pigs, carp, catfish, salmons, shrimps, tilapias, and trout ,24. From aureus) . Then, b2This section provides an overview of our method, followed by the data we collected and analyzed.2.1Non-spatial regression models assume that observations are independent , but thiSpatial econometric methods have been applied to various health conditions, both communicable and noncommunicable, in a growing body of literature . However(1)To explore the effect of AMU in farmed animals on AMR in humans in a country.(2)To assess how policy actions in one country can influence the AMR situation in neighboring countries.(3)To examine if AMR can spread over the national borders.We employed a Spatial Durbin Model (SDM) since it can address our three main objectives:The SDM is a spatial econometric model incorporating the Spatial Autoregressive model (SAR) and the Spatial Lag model (SLX) features. It assumes that the dependent variable is influenced by its spatial lag and the spatial lags of the independent variables. Less intuitively, the SDM can also incorporate the Spatial Error Model (SEM), which accounts for the spatial dependence among the error terms . The stat is the time). StataSE17, which uses the Quasi–Maximum Likelihood (QML) method for fitting spatial panel models.Where ocations ,36. Spatelements . ui is a2.2This section first outlines the scope of the study, which covers our sample and the timeframe. Next, we define the variables and data that we used for the modeling, including the data's sources, measurements, and transformations.2.2.1We used publicly available data to determine the largest animal producing countries in each region of the world  for cattle, chickens, pigs, and aquatics . We collected data on production (2010 to 2020) from the online database of the Food and Agriculture Organization of the United Nations (FAO). Figs. A1 to A4 in Appendix A show the selected producer countries and their related share in global production. The selected countries were thirty and produced 51% of the world's cattle, >60% of chickens, >70% of pigs, and >90% of aquatics. They can also be considered the largest consumers of veterinary antibiotics.2.2.2E. coli and S. aureus in humans was calculated as the ratio between the number of resistant bacterial isolates and the total number of isolated tests (Eq. 2) and included in the model as the dependent variable (named AMR in humans).The Multi-Antibiotic Resistance (MAR) index  for E. cE. coli and S. aureus's antibiotic resistance were collected from the Center for Disease Dynamics, Economics and Policy (CDDEP) online database [Data on database . Since tdatabase , we builR is resistant to antibiotic.N is the number of the isolated tests.n is the number of antibiotics.2.2.3misuse and overuse of antimicrobials; lack of access to clean water, sanitation, and hygiene (WASH) for both humans and animals; poor infection and disease prevention and control in healthcare facilities and farms; poor access to quality, affordable medicines, vaccines, and diagnostics; lack of awareness and knowledge; and lack of enforcement of regulation” [While there are numerous contributing factors to AMR, we used the WHO's definition of primary causes of AMR in humans to determine the independent variables for the analysis. According to the WHO, the main drivers of AMR include “ulation” .(1)misuse and overuse of antimicrobials: AMU in humans and AMU in farmed animals. We obtained the data regarding AMU in humans from the online database of CDDEP and estimated (2016–2020) using a linear regression [We defined two variables for gression . Regardigression . For aqugression ,23,24 fogression ,45.(2)A dummy variable was created to represent the poor access to WASH infrastructure, using “effect coding(3)We considered the percentage of GDP used on health expenditures in a country as a representative of poor infection and disease prevention and control in healthcare facilities and farms, and poor access to quality, affordable medicines, vaccines, and diagnostics. The source for this data is the World Bank .(4)We added another dummy variable to the model to evaluate the effect of management policies at the farm level in a country on AMR in humans in that country and neighboring countries. This dummy serves as an indicator of a lack of regulation. One strategy to minimize the AMU in farmed animals is to restrict or reduce the use of antibiotics as growth promoters . In thisBased on the above definition, we have defined the following independent variables to use in our empirical model:The descriptive summary of the variables is reported in 3p-value = 0.002) close to zero indicates that data are correlated across panel groups. In modeling terms, this behavior displays a significant coefficient on the spatially lagged dependent variable. The outcome allowed us to follow spatial models. Then, a spatial weight matrix was prepared using GeoDa software and used in Stata. The matrix is 30 × 30, row-standardized with zero diagonal factors, and developed via the first-order queen contiguity. The Pesaran-CD test  was perf2 = 3.20; p-value = 0.6690). Parameter ρ in Partial deviations afford an incomplete understanding of how the independent variables affect the dependent variable in spatial panel models. Henceforth, marginal effects have to be taken into account, which consist of three components: direct, indirect, and total effects. The direct effect measures how a change in an independent variable in one place affects the dependent variable in the same place. The indirect effect measures how a change in an independent variable in one place influences the dependent variable in the neighboring places. The total effect is the sum of the direct and indirect effects. Direct effects are well estimated by the SDM spatial model, even when the model is not correctly specified. However, the model tends to overestimate indirect effects . We procmisuse or overuse of antibiotics can contribute to AMR in humans. For a 1% increase of AMU in farmed animals, AMR in humans increases by 0.04%. Similarly, AMU in humans is also positively correlated with AMR in humans, which means that higher levels of AMU in humans lead to higher levels of AMR. For a 1% increase of AMU in humans, AMR in humans increases by 0.19%. Our analysis revealed unexpected evidence about the signs of indirect effects. A solution to this issue is increasing the sample size. Regarding their values, it should also be taken into account that the SDM suffers from an overestimation of indirect effects.As shown in To robustly support our results, we have also evaluated the spatial models on AMR in humans for the 28 EU countries . Due to the unbalanced panel structure of the data,4Even though most antibiotics sold worldwide are used for farmed animals, AMR in humans related to AMU in farmed animals has been understudied and remains unknown. Additionally, although many studies warn about the transmission of AMR between humans, animals, and the environment and that AMR can cross countries' borders, statistical models have not yet confirmed this spreading.E. coli and S. aureus). The model does not analyze factors that may influence AMR in humans comprehensively. Nonetheless, it focuses on critical variables based on the main drivers of AMR indicated by the WHO.Using spatial analysis, in this study, we explore the connection between AMU in the major farmed animal species and AMR in humans related to two of the most lethal bacteria , while E. coli resistance to Glycylcyclines, banned in animal farming, is 0.78% (very low resistance). For S. aureus, resistance to Macrolides represents 56.0%, considered very high, while resistance to Vancomycin, a more recent antibiotic banned in animal farming, is very low (0.22%). This evidence shows the importance of avoiding the use of critical antibiotics for both humans and farmed animals.WHO claims that DEP data , the nonoveruse of antibiotics in humans and animals. Based on a study in the United Kingdom [Another primary cause of AMR in humans, confirmed by the WHO, is the  Kingdom , cattle  Kingdom . TherefoLastly, the estimated model in this study showed a significant spatial dependence . The model's most relevant independent variable for the analysis is the level of AMU in farmed animals by species. We needed a ten-year historical series from the selected countries for this variable. Such data were available only for France, Denmark, the United Kingdom, and the United States. For the other selected countries, we calculated the total AMU based on FAO data on animal production in the different countries multiplied by the global averages of antibiotic consumption for the various species estimated by other authors ,23,24. TWe recommend future studies to apply the same models to a more countries with different production and consumption levels and roles. Moreover, the models should incorporate a weight matrix that captures the various dimensions of connectivity among countries, such as trade, travel, and other forms of interaction, rather than relying solely on distance.5misuse and overuse prevention) in animal farming and harmonize practices in an integrative approach . Examples of husbandry systems that phased out antibiotics suggest that strict limitations on non-therapeutic uses are possible with only minor reductions in productive performance and animal health.This study found a positive association between AMU in farmed animals and AMR in humans using a spatial econometric model. Additionally, the model showed the relevance of AMR spread between countries. Therefore, although the use of antibiotics in farmed animals is currently under control in some countries, many others still need to enforce laws in this area, allowing for unfavorable cross-border effects at the regional and global levels. Health issues such as AMR reveal the interconnectedness of living beings and their shared ecosystems. Thus, more severe actions are necessary at national, regional, and global levels to optimize AMU (Zahra Ardakani: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Massimo Canali: Funding acquisition, Investigation, Project administration, Supervision, Validation, Writing – review & editing. Maurizio Aragrande: Conceptualization, Methodology, Validation, Writing – review & editing. Laura Tomassone: Conceptualization, Methodology, Validation, Writing – review & editing. Margarida Simoes: Conceptualization, Methodology, Validation, Writing – review & editing. Agnese Balzani: Writing – review & editing. Caetano Luiz Beber: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – review & editing.10.13039/501100005969University of Bologna. One of the authors is an employee of World Animal Protection. World Animal Protection did not have any role in the study design, collection, analysis, and interpretation of data.World Animal Protection provided financial support for the research to the Department of Agricultural and Food Sciences of the


After spinal cord injury (SCI), endoplasmic reticulum (ER) stress has been reported to be an integral part of the secondary injury process that causes apoptosis of glial cells, leading to remyelination failure. This report focuses on exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist widely used to treat diabetes, as a potential agent to improve functional outcome after SCI by improving the ER stress response. Exenatide administered subcutaneously immediately after injury and 7 days later in a rat model of moderate contusive SCI revealed significant improvement in hindlimb function without any hypoglycemia. Changes in the expression of glucose regulatory protein 78 (GRP78), an endoplasmic reticulum chaperone that protects against ER stress, and C/EBP homologous transcription factor protein (CHOP), a pro-apoptotic transcription factor in the apoptosis pathway were examined as indices of ER stress. We found that administration of exenatide after SCI suppressed CHOP while increasing GRP78 in the injured spinal cord, leading to a significant decrease in tissue damage and a significant increase in oligodendrocyte progenitor cell survival. This study suggests that administration of exenatide after SCI decreases ER stress and improves functional recovery without any apparent side-effects. Spinal cord injury (SCI) is a devastating condition that leads to life-long physical and mental distress, and there is currently no established treatment. The pathophysiological events of traumatic SCI are twofold: the primary injury resulting from the direct physical impact, and the subsequent secondary injury that occurs as a consequence of the primary injury and expands the lesion area due to biochemical and vascular sequelae . Recent When cells encounter stress such as electrolyte imbalance or increased excitatory amino acids, the normal folding of translated proteins within the ER is disrupted. This leads to the accumulation of unfolded or misfolded proteins in the ER in a condition that has been aptly termed ER stress . This trThe cellular response and unfolded protein response to this ER stress have been reported in SCI , but theExenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist widely used as an antidiabetic drug, has also been reported to reduce ER stress in multiple systems such as the pancreas beta cells, joint chondrocytes, and hyperglycemic cardiomyocytes and has been effective in treating amyotrophic lateral sclerosis (ALS), multiple sclerosis, and traumatic encephalopathy . AlthougAll animal procedures were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee at Tokai University School of Medicine. Female Sprague-Dawley (SD) rats  were purchased from Nippon Crea  and used for the experiment. Surgery was performed with 4% isoflurane inhalation anesthesia under aseptic conditions. After laminectomy of the 10th thoracic vertebra, the dura was exposed, and a contusive SCI was induced with a force of 200 Kdyne and dwell time of 0 s using an Infinite Horizon spinal cord impactor device . The actual force applied to the dura mater was recorded and compared among the exenatide and control groups, confirming that there was no statistical difference between the two groups. Bladder massages were performed twice a day until voluntary urination was restored.The injured rats were divided into three groups: exenatide, control, and sham groups (n = 10 per group). The exenatide group received 10 µg subcutaneous injection  immediately after injury and at day 7 after injury, while the control group received phosphate buffered saline (PBS) injection and the sham surgery group received a laminectomy without a spinal cord contusion procedure.Hindlimb motor function was evaluated using the Basso, Beattie, and Breanahan locomotor rating scale  before injury, the day following surgery, and every other day until day 14 after surgery. The evaluation was performed between 9 am and 12 noon by 3 researchers blinded to the procedure, and the mean values of 5-minute observations were used (n = 10 per group).Blood glucose levels were measured immediately before the injury, immediately after the injury, 3, 6 and 12 h after the injury and every other day thereafter (n = 10 per group). Approximately 1 ml of blood was collected from the caudal vein using a 20 G needle, and blood glucose was measured using a Medisafe FIT blood glucose meter .The injured spinal cord was exposed under isoflurane 4% inhalational anesthesia on days 1, 3, 7, and 14 after injury, and a 5 mm section of spinal cord tissue centered on the damage epicenter was resected under a microscope. Immediately after removal, the spinal cord was washed in ice-cold PBS, and processed with Cell Lytic NuCLEAR extraction kit . 20 µg of spinal cord lysate was loaded into each lane of a 10% SDS polyacrylamide gel, subjected to electrophoreses, and electrotransferred onto nitrocellulose membranes . The membranes were blocked with 5% BSA in TBST , then incubated overnight at 4 °C with rabbit anti-GRP78  and rabbit anti-CHOP  antibodies. The membranes were washed in 0.05% Twin-20 in PBS for 7 h, then incubated for 60 min at 25 °C with horse radish peroxidase (HRP)-linked anti-Rabbit IgG . The proteins were labeled with Immobilon Western Chemiluminescent HRP  and analyzed using densitometric scans of the films using the Macintosh software CS analyzer  and normalized against the internal control β-actin labelled with mouse monoclonal antibody against β-actin . Expression of GRP78 and CHOP over time was compared between the exenatide group and the control group (n = 5 per group) and presented as a ratio of the mean expression observed in sham animals.Perfusion fixation was performed using 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) under general anesthesia with isoflurane 4% on days 3, 7, and 14 after injury. After perfusion fixation, the spinal cords were removed and fixed in 2% PFA in 0.1 M PB at 4 ℃ for 2 days, followed by a dehydration process in stages with 7%, 15% and 20% sucrose water. The spinal cords were embedded and frozen in Optimal Cutting Temperature (OCT) compound , and were sectioned at a thickness of 10 µm on a cryostat microtome . A 2 mm area equating to the tip of the IH impactor was set as the epicenter, and sections were prepared at 3 mm intervals, at the epicenter, 4 mm, 7 mm, 10 mm and 13 mm.Day 14 sections were stained with hematoxylin-eosin (HE) and Luxol fast blue (LFB) (n = 5 per group). For HE staining, sections were washed in deionized water for 5 min and then continuously stained by hematoxlin for 2.5 min and by eosin for 16 s. For Luxol Fast Blue (LFB) staining, sections were stained with a 0.1% LFB (Sigma-Aldrich) solution, sealed, washed with distilled water, and placed in 95% alcohol for 10 min. Each mixture was separated in a 0.05% lithium carbonate aqueous solution for 10 s and in a 70% alcohol solution for 20 s. The above two steps were repeated until the grey and white matter were clearly observable under the microscope. The above sections were dehydrated by a conventional alcohol gradient , placed in xylene I and II for 10 min, and then sealed with neutral gum. Images were captured with an Olympus Microscopy Europa BX63 , and the lesion cavity and myelinated areas were quantified with cellSens Dimension . The cavity volume was estimated utilizing Cavalieri’s principle, in which the cavity area on each section was multiplied by intersection distance and totaled into a series summation. For the evaluation of LFB-positive myelinated area, the average of three sections per animal were evaluated and presented as a percentage of the total area.Sections taken 7 mm caudal to the lesion epicenter from spinal cords collected on day 3, 7 and 14 after injury were used for cytological immunohistochemical staining (n = 5 per group). The lesion epicenter was specifically not selected because the damage at the epicenter led to significant loss of cells that made quantification difficult. The sections were washed 3 times for 10 min with PBS, blocked at 24 ℃ for 60 min with 5% normal goat serum in PBS, washed again for 10 min, and then incubated overnight at 4 ℃ with rabbit anti-GRP78 , rabbit anti-CHOP , mouse anti-NG2 , and mouse anti-GFAP. The sections were washed with PBS, then incubated in a darkened room for 60 min at 24 ℃ with the following secondary antibodies: goat anti-rabbit Alexa Fluor 594 , goat anti-mouse Alexa Fluor 488 . VECTASHIELD with DAPI (Vector Laboratories) was used for nuclear staining and encapsulation and the stained sections were observed with a fluorescence microscope. The total number of NG2-positive OPCs and GFAP-positive astrocytes in the dorsal column of five consecutive sections were counted. The ratios of these cells that were also positive for GRP78 or CHOP were calculated, and the differences between the two groups were compared (n = 5 per group).All data were analyzed using two-way repeated-measures analysis of variance (ANOVA) with Tukey’s post hoc multiple comparison tests. The level of significance was set at p < 0.05.The blood glucose level, which is normally approximately 120 mg/dL in rats, increased to approximately 200 mg/dL in both the exenatide and control groups immediately after injury, but quickly returned to normal levels thereafter. There was no significant difference observed between the two groups and no hypoglycemia was observed in the exenatide group .Fig. 1ExThe effect of spinal cord injury on ER stress and the effect of exenatide administration were examined by studying the expression of GRP78 and CHOP by western blot. The levels of GRP78, the ER chaperone of the unfolded protein response that enhances the cell’s mechanisms to lower ER stress, were higher in the exenatide group on higher on days 1 and 3 and lower on days 7 and 14, with a significantly higher expression on day 3 after injury (p < 0.05) . The levThe lesion cavity volume was evaluated with HE-stained sections from day 14 animals, revealing a significantly larger cavity in the control group compared to the exenatide group . MyelinaCell-specific expression of the ER-stress indices was examined by immunohistochemistry. NG2-positive oligodendrocyte precursor cells and GFAP-positive astrocytes in the dorsal column that were also positive for GRP78 or CHOP were quantified. GRP78 immunoreactivity throughout the entire dorsal column tissue tended to be higher in the exenatide group at all time points, with a statistically higher ratio of positive cells in both OPCs and astrocytes of the exenatide group compared to the control group at day 3 (p < 0.01) , Fig. 7.The BBB scale was 0 on the day after injury in both groups and subsequently improved over time. The exenatide treatment group showed a significant improvement in BBB score from day 7 onwards compared to the control group (p < 0.05), with the mean BBB scale at final observation on day 14 after injury being 15.4 in the exenatide treatment group while it was 8.9 in the control group .Fig. 11HL-cells to activate insulin production in pancreatic β-cells and lower blood glucose levels. GLP-1 functions by activating its specific receptor, the GLP-1 receptor (GLP-1R), and activation of the GLP-1R has been shown to have a wide range of effects, such as neuronal cell proliferation and differentiation, neurite outgrowth, and enhancement of synaptic plasticity  from apoptosis, leading to an increased number of oligodendrocytes that can remyelinate neurons and improving hindlimb motor function. Contusive SCI has been shown to be predominantly a white matter injury, and we have focused our efforts on the oligodendrocyte lineage. This study is a preliminary exploratory project undertaken in order to probe whether or not exenatide affected ER stress levels within the injured spinal cord, and we posit that exenatide does in fact reduce ER stress. At the same time, it is important to acknowledge that SCI is a traumatic event that involves a wide variety of cellular stresses that activates the integrated stress response in which the ER stress response is a part of. We are currently performing follow-up studies looking into the molecular pathways involved in the ER stress response.The involvement of ER stress in SCI as well as the changes seen in the three signal pathways Perk, ATF6, and IRE1a of the unfolded protein response has been well studied . In thisThere are several limitation to this study. The greatest limitation stemmed from the short study period. This study was conducted to examine the short-term effects of exenatide administration on the recovery from SCI and has demonstrated significant molecular, histological, and behavioral improvements in the exenatide group compared to the control group. One limitation is the lack of multiple behavioral assessment tests. While the difference observed in hindlimb motor function is significant, the improvement would be more convincing if motor or sensory improvements were confirmed with another functional test. Another major limitation of this study is in the follow-up period of 14 days. The BBB data over the 14 days was not of sufficient length to follow the hindlimb motor function improvement from SCI until the animals of the two injured groups reached a plateau. Therefore, we cannot confirm if improvements seen in the exenatide group were maintained in the chronic phase after SCI. The study period was also not of sufficient duration to study the effects of exenatide on the oligodendrocyte lineage or its involvement in remyelination. This was an exploratory study to verify that exenatide administration after spinal cord injury improves motor function after SCI without severe side effects, and to research the possible involvement of ER stress in this process. We believe that the study period is sufficient to demonstrate that exenatide modulates ER stress, but its specific effects on the multiple pathways involved in the regulation of the UPR as well as the effects of this decreased ER stress will require longer study periods. Another limitation concerns our interpretation of the effect of exenatide on OPCs. We interpreted the increase of OPCs in the exenatide group as evidence of increased survival of the proliferating OPCs, but in fact we cannot exclude the fact that exenatide administration could increase the proliferation of OPCs or affect differentiation from local neural stem cells to favor the oligodendrocyte lineage. With the results of this study demonstrating significant improvement, our next step will be to study the long-term effects of exenatide as well as the dosage and administration schedule of exenatide.Combining the results of this study with previous reports on ER stress, it is thought that exenatide reduces secondary damage in the injured spinal cord by decreasing ER stress, thereby suppressing apoptosis, protecting myelin sheathes, and improving hind limb function after SCI. Past reports of exenatide administration after SCI have demonstrated that exenatide has multiple beneficial effects. The collective data indicate that exenatide enhances the mechanisms that regulate cell homeostasis, such as ER stress, mitochondrial stress, and autophagy, resulting in a net reduction in the secondary injury process. Further studies are needed to examine the full effects of exenatide on the ER stress response in the injured spinal cord as well as its effects on different cell types, including macrophages, mature oligodendrocytes, and neurons.The Institutional Review Board of the Tokai University.10.13039/501100001691JSPS KAKENHI Grant-in-Aid for Scientific Research (C) number 18K090984 and 20K09417.This work was supported by Satoshi Nomura: Conceptualization, Methodology, Validation, Formal analysis, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization,. Hiroyuki Katoh: Conceptualization, Methodology, Writing – review & editing. Sho Yanagisawa: Methodology. Toshihiro Noguchi: Validation, Visualization. Keiko Okada: Validation, Visualization. Masahiko Watanabe: Project administration.The authors have not competing interests to declare.


Kaposi sarcoma (KS) of the gastrointestinal (GI) tract in a patient with acquired immunodeficiency syndrome (AIDS) has not been reported in an adolescent outside of Africa. We present a 16-year homosexual old male with AIDS, cutaneous KS, pulmonary KS, and gastrointestinal KS (GI-KS) lesions. Eighty percent of patients with GI-KS are asymptomatic, but our patient presented with a month-long history of dysphagia, abdominal pain, and hematochezia. Endoscopy with biopsies revealed multiple KS lesions within the stomach and lower GI tract. This novel case demonstrates the importance of considering early endoscopic screening in immunocompromised adolescents with cutaneous KS to improve morbidity and mortality. Kaposi sarcoma (KS) is a rare malignancy caused by the human herpes virus 8 (HHV-8) infection. This angioproliferative tumor most often manifests as multifocal red to purplish cutaneous plaques. Four main types of KS have been identified: classic, endemic, iatrogenic, and AIDS-associated. In the United States, acquisition of KS lesions are most commonly associated with AIDS . The incA 16-year-old homosexual male with a history of asthma who was sexually active presented with multiple cutaneous KS lesions and was found to have an initial human immunodeficiency virus (HIV) RNA viral load of 59,059 copies/mL and a CD4 antigen positive cell count of 58 cells/μL. The patient’s diagnosis of HIV-AIDS with cutaneous KS was made at that time, and the patient was started on daily ART. Baseline endoscopy and colonoscopy were not performed. Despite ART therapy, the patient reported a month history of dysphagia and generalized abdominal pain. In addition, he described several months of watery diarrhea that progressed to hematochezia.Two months after his initial diagnosis of HIV-AIDS with cutaneous KS, he presented to the emergency department (ED) with respiratory distress, abdominal pain, and hematochezia. In the ED, the physical exam was significant for multiple violaceous plaques on face, chest Fig. , back, aOver the next 4 days, he stabilized and further evaluation of his gastrointestinal symptoms ensued. The case was reviewed with the NIH. Before proceeding with chemotherapeutic treatments, the NIH recommended a bronchoscopy, and an upper and lower GI endoscopy due to concern for visceral KS manifestations. He continued to have minimal mucus discharge from his rectum, stool hemoccult was positive, and a stool infectious work-up was negative. Abdominal CT was significant for an ill-defined, thick-walled appearance of the cecum and terminal ileum.The upper endoscopy revealed a grossly normal esophagus and duodenum with areas of mild erythema. In contrast, the entire gastric mucosa was covered with superficial maculopapular lesions and multiple polypoid masses—the largest mass was in the gastric fundus Fig. . Due to In the stomach, cecum, sigmoid, and rectum, the histologic examination revealed findings consistent with GI-KS Fig. A—confirm2). His chemotherapy was adjusted in both dosing and agents based on his clinical response. Despite continued medical care and close follow-up, he died 8 months later due to disease progression and opportunistic infections.The patient continued the daily ART regimen, and started IV liposomal doxorubicin (20 mg/mA 2014 Cochrane review, “Treatment of KS in children with HIV-1 infection,” examined 4 pediatric cohort studies from individual African countries, as pediatric data are sparse outside Africa. In a disease with a significant risk of morbidity and mortality, the most fortuitous outcomes of KS remission and decreased mortality risk were observed in patients receiving the combination of ART and a chemotherapeutic regimen, in comparison to each treatment alone. However, insufficient pediatric data exists to adequately evaluate the efficacy of these 4 differing chemotherapeutic treatment protocols .In AIDS-KS patients, the GI tract is the most common site for extracutaneous KS lesions with an incidence greater than 50% ,6,10. ThUltimately, treatment regimens rely on the TIS system, as patients with localized disease tend to have better outcomes when compared with more advanced staging. In immunocompromised patients with cutaneous KS, these multiple factors demonstrate the importance of endoscopic screening to enhance early detection and guide treatment in an effort to improve overall morbidity and mortality ,11.All attempts have been exhausted in trying to contact the parents or guardian for the purpose of attaining their consent to publish the Case Report. All identifying information has been removed from this case report to protect patient privacy.