The chemical structure of the gel beads was assessed using FTIR spectroscopy, while their thermal stability was evaluated through differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Due to the alginate matrix, the composite Alg/ZA5-Cur beads possess pH-responsive properties. In addition, the gel beads were modified by chitosan (CS) to enhance the stability and control the degradation behavior of the gel matrix. The swelling behavior and the degradation of the beads were analyzed in physiological solutions with different pH values. Results demonstrate the stabilizing and protective effect of the chitosan coating, as well as the reinforcing effect of the zeolite filler. This makes the pH-responsive alginate gel beads good candidates for the delivery of lipophilic drugs to specific inflammatory sites.Alginate-based beads containing a porous zeolite filler were developed as carriers of bioactive compounds with a hydrophobic nature, such as curcumin (Cur). Curcumin, a natural pigment extracted from the turmeric (Curcuma longa) plant, exhibits antioxidant, anti-inflammatory, anticarcinogenic, and antiviral properties. To enhance the bioavailability of the drug, curcumin needs to be encapsulated in a suitable carrier that improves its dispersibility and solubility. Commercial A-type zeolites (Z5A) were used as curcumin-binding agents and they were immobilized within the alginate gel beads by cross-linking in calcium chloride solution during an extrusion dripping process. The process parameters (alginate and CaCl Drug delivery systems play a crucial role in ensuring efficient and targeted delivery of therapeutic agents ,2. In reAlginate-based gels in the form of beads, microspheres, and nanoparticles are widely utilized in the biomedical field because they provide precise control over drug release rates and enable specific drug delivery to targeted therapeutic sites . Additio2+ ions, alginate can form a water-resistant gel network via ionotropic gelation at room temperature, allowing immobilization under mild and safe conditions. Alginate beads can encapsulate bioactive compounds, including phenolic compounds [Alginate is a natural anionic linear polysaccharide composed of alternating blocks of α-L-guluronic and β-D-mannuronic acid residues linked by 1–4 bonds. Through intermolecular cross-linking with divalent cations such as Caompounds , lipophiompounds , and proompounds . After dpH-responsive alginate beads have great potential for delivering and controlling release of fat-soluble active principles, like curcumin. Curcumin is a polyphenolic compound isolated from the rhizome of the herb Curcuma longa. It exhibits low intrinsic toxicity and high therapeutic efficacy but suffers from challenges such as low water solubility, stability, and poor oral bioavailability, which limit its applications. Sreekanth Reddy et al.  fabricatIn some works, curcumin is loaded into inorganic materials, such as zeolites, to improve its stability. Zeolites are microporous inorganic crystalline materials with a three-dimensional framework comprising silicon, aluminum, and oxygen. They possess several advantages, including a well-defined pore structure, tunable base-acid sites, molecular sieve properties, and ion exchange capabilities. These materials are successfully used as carriers of various therapeutic compounds  and are This work introduces a promising approach for the transport of lipophilic drugs, which cannot be solubilized in aqueous media, using composite alginate/zeolite gel beads in which the active compound is immobilized within the zeolite porous structure. In fact, curcumin is a lipophilic molecule that is not directly soluble in the highly hydrophilic alginate beads. For this reason, in this work, curcumin was first loaded in porous zeolites before integration with the alginate-based gel carriers. An l-type zeolite molecular sieve (zeolite A) was employed as a drug binder for curcumin and dispersed in a sodium alginate solution to prepare pH-sensitive composite beads for controlling the release of curcumin. The alginate beads were fabricated using ionic gelation with extrusion-dripping method and the process parameters were optimized. The beads coated with chitosan were also prepared, to improve the response to pH. As reported in the literature ,23,24, tThe alginate-based gel beads are extensively characterized using various techniques, such as optical microscopy, Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). These characterization techniques provide insights into the structural morphology, chemical composition, and pH-responsive behavior of the gel beads, ensuring their suitability for drug delivery applications.The synthesized gel beads offer pH-responsive release properties, enhanced stability, and improved therapeutic efficacy. This research holds significant potential for the development of advanced drug delivery systems with applications in various biomedical fields.The fabrication process for Alg/Z5A/Cur beads was developed and optimized using the extrusion-dripping technique through ionic gelation. This process is schematically represented in 2 solution , the collecting distance  and the needle gauge . Results are summarized in 2 solution increased. This can be attributed to the shrinkage of the gel network as the concentration of Ca2+ ions increased [2 solution show an irregular, non-spherical morphology due to the high surface tension of the gelling bath during impact.In preliminary tests, alginate beads were prepared by varying the concentration of alginate , the concentration of the CaClncreased , leading2 cross-linking solution. Each parameter was individually varied to assess its effect on the samples.The best results, in terms of size and shape, were achieved using a 2 wt% alginate solution and a 10 wt% CaClThe collecting distance is an important parameter that strongly depends on the viscosity of the alginate solution. Low-viscosity alginate solution droplets tend to flatten upon impact with the crosslinking bath, even at short collecting distances, which can lead to non-spherical beads. In this study, four different collecting distances were tested: 1.6 cm, 7 cm, 10 cm, and 11.5 cm. 2 cross-linking solution, and a collecting distance of 7 cm.The stirring speed during the polymerization process was investigated, and it was found that a stirring speed of 200 rpm resulted in elongated beads. As a result, the stirring speed was set to 0 rpm. The best results, in terms of size and shape, were achieved using a 2 wt% alginate solution, 10 wt% CaCl3+ from chitosan and COO− from alginate form an ionic bond [The chitosan coating was applied to the beads by transferring them into a chitosan solution and vortex-mixing the suspension at 800 rpm. During the coating process, the functional groups NHnic bond . The effThe weight and diameter of beads before and after coating were compared, for each of the tested speeds. The results are listed in The value of 800 rpm was selected for the preparation of small-size beads. Indeed, few works in the literature report that the coating applied through stirring may induce the shrinkage of beads ,22. In oFor the fabrication of the composite beads, the alginate solution containing Z5A/Cur was dripped into the cross-linking solution. The process parameters used were those optimized in the preliminary experiment.The dry beads shown in Dimensional analysis and sphericity factor (SF) evaluation were conducted on swollen and dried beads using optical microscopy. The mean diameter values and SF of the beads are reported in p < 0.05). Additionally, the sizes of the beads decrease significantly upon drying, which can be attributed to their high-water content, as shown in p = 0.0177). All types of beads have an SF ≤ 0.05, indicating their close approximation to a spherical shape, except for the dry alginate spheres with and without coating. The use of spherical beads might have several advantages. The spherical shape of the beads can provide a more uniform release of the drug, which occurs through surface erosion of the gel beads. This might facilitate the control of the drug release over a defined period, thus improving therapeutic results. In addition, this geometry maximizes the ratio of volume to surface area resulting in increased loading capacity. Finally, the symmetrical nature of the spherical beads increases their structural strength, reducing the risk of deformation or breakage along one direction during handling.For all types of hydrated beads, the modification by chitosan (at a vortex stirring rate of 800 rpm) results in a statistically significant decrease in diameter when compared to the pure alginate beads . The TGA curve of Z5A indicates a mass loss that can be attributed to the release of water trapped in the nano-porous zeolites. Z5A samples loaded with Cur exhibit similar behavior to that of pure zeolites. Additionally, above 250 °C there is a mass loss associated with the thermal degradation of curcumin. The thermogram of the Alg/Z5A/Cur beads shows three distinct thermogravimetric effects. In the low-temperature range (50–180 °C), an initial mass loss occurs due to the desorption of water trapped in the zeolite cavities and water bound to the alginate. In the intermediate temperature range (180–300 °C), a mass loss is observed, which can be attributed to the breakdown of the alginate backbone and the thermal degradation of curcumin. In the higher temperature range (300–550 °C), mass loss occurs due to the thermal degradation of both alginate and curcumin. The curve of the Alg/Z5A/Cur/CS beads exhibits a similar trend to those without chitosan coating. However, there is a higher mass loss compared to the previous case, as indicated in Differential scanning calorimetry (DSC) on pure zeolites, Z5A/Cur, and Alg/Z5A/Cur beads, with and without chitosan coating was performed in the temperature range 50–270 °C. The thermograms are reported in −1 and a band at 1636 cm−1 corresponding to the stretching of O–H and asymmetric stretching vibration of alginate COO groups, respectively. The antisymmetric stretching of the alginate carboxyl group (1420 cm−1) and the stretching vibrations of C– OH side groups (1032 cm−1) can be observed in alginate beads. In alginate beads with chitosan coating, the peaks at 1464 cm−1 and 1366 cm−1 are caused by the deformation of the C– H and CH2OH groups of chitosan [−1 falls within the band of aliphatic ethers and could be attributed to the antisymmetric stretching of the C–O–C bridge [chitosan . The peaC bridge . The pre−1 and 2852 cm−1, which are due to C–H stretching [−1 is attributed to the stretching of the C=O group of curcumin [−1 and 1162 cm−1 is attributed to the vibration of the C–H group and the deformation of the aromatic ring. In the fingerprint zone, the stretching vibration of the Si–O–Al bridge at 669 cm−1, typical of zeolites [−1 is due to the symmetrical deformation of methyl groups of chitosan, and the peaks at 1246 cm−1 and 1158 cm−1 are attributable to the skeletal deformation (C–CHO), probably superimposed on the C–O stretching of the aromatic ring of curcumin [−1 is caused by the overlapping of the C–O and C–O–C stretching of alginate and chitosan [The spectra of composite beads show peaks at 2939 cmretching  and the retching , respectcurcumin ,32. The zeolites , confirmcurcumin . The abschitosan .Swelling kinetics and degradation tests were conducted to evaluate the pH response of the beads and the stabilizing effect of chitosan coating. A preliminary test was conducted on empty, filled beads with and without chitosan coating immersed in a PBS solution with a pH of 7.4 at room temperature (25 °C) for 6 h . All typIn addition, the empty beads exhibit a higher swelling ratio compared to the loaded ones; this could be attributed to the zeolites encapsulated within the three-dimensional structure of the gel, which limit the extension of the polymer chains ,38. To assess the effect of the pH value on the swelling ratio, swelling kinetics was performed on Alg/Z5A/Cur beads with and without chitosan coating in simulated gastric fluid , acid tumoral environment (pH = 5.6), simulated intestinal fluid  and simulated colon fluid  for 6 h at 37 °C. a ), the concentration of -COO− increases, increasing electrostatic repulsion between the carboxylate anions and the swelling ratio [3+) as the pH < pKa (CS), so degradation does not occur : the interaction between the two polysaccharides is weak and the swelling increases due to the increase of the electrostatic repulsion. Degradation at pH = 7.4 is shown in At pH = 6.8, the deprotonated carboxyl groups increase, and the protonated amines decrease. In Alg/Z5A/Cur/CS beads, the interaction between chitosan and alginate decreases and the swelling increases due to the electrostatic repulsion of -COOComposite alginate/zeolite gel beads with pH-responsive properties were developed as carriers for lipophilic drugs, such as curcumin. The drop extrusion technique was used to crosslink the beads’ polymer network and the process parameters were optimized. The properties and the swelling behavior of the composite beads were studied using various methods. FTIR spectroscopy verified Z5A/Cur incorporation in the alginate network. Optical microscopy showed round gel beads (SF < 0.05) with a compact structure, smaller when chitosan-coated due to electrostatic effects. Swelling ratio analysis indicated high water content (>96%), which is compatible with soft tissue microenvironments. Thermal analysis  confirmed curcumin loading in zeolites. The Alg/Z5A/Cur gel beads demonstrated different degrees of swelling and stability depending on the environment pH, with lower swelling in acidic conditions, such as in simulated gastric fluid (SGF). Beads remained stable in acidic environments and started degradation at pH 5.6. The chitosan modification further improved stability of the beads in an acidic environment. Results in terms of pH-responsive behavior and stability make Alg/Z5A/Cur/CS beads promising carriers for lipophilic drugs in specific inflammatory sites.2) dihydrate, acetic acid, curcumin, and phosphate buffered saline were purchased from Sigma-Aldrich . Zeolite 5A, the calcium-exchanged form of zeolite Linde type A, was obtained in bead form from Zeochem AG . Ethanol was purchased from VWR Chemicals . Deionized water (resistivity 18.2 MΩ⋅cm) was produced by a Direct-Q3 UV water purification system  and used in all preparations.Alginic acid sodium salt, chitosan, calcium chloride , collecting distance , and needle gauge . The chitosan-coated beads were prepared by vortex mixing in a 0.5 wt% chitosan solution for 30 min, then rinsed and stored in deionized water. Three different mixing speeds were evaluated during the coating process: 0 rpm, 300 rpm, and 800 rpm.Sodium alginate was added to ultrapure water at 1–4 wt% concentrations and mixed for 2 h using a magnetic stirrer . Beads were produced using the extrusion-dripping method, where the prepared alginate solutions  were added dropwise using a 1 mL syringe into a CaClCurcumin was loaded in the porous zeolites following a previously published protocol . BrieflyAn amount of 0.2 g of Z5A-Cur was added to 10 mL of alginate solution to prepare the composite beads. The Alg/Z5A/Cur solution was stirred for 15 min and then sonicated for 45 min to achieve a homogeneous solution.2 aqueous solution, following the parameters optimized in the initial steps of the work. The beads were further optimized with a chitosan coating.Composite beads were fabricated using the extrusion-dripping method, where the Alg/Z5A/Cur solution was added dropwise into a CaCl1D represents the larger diameter of the beads, while 2D is the smaller one. When SF ≤ 0.05, the shape of the matrix can be approximated to a sphere. Data were analyzed using a t-test to determine any statistical difference. The significance was determined at the 95% confidence level.A Leica DMLP polarized microscope equipped with 5×, 10×, and 20× objective lenses was utilized to analyze the surface morphology and particle size of the beads. The average size of the beads was measured using ImageJ software (version 1.53). The sphericity factor (SF) was determined using the following equation :(1)SF=D1The thermal properties and thermogravimetric analysis (TGA) of zeolite, curcumin-loaded zeolite, and composite bead samples were investigated using a TGA/DSC 1 STARe instrument . For the thermal analysis, dry samples weighing approximately 10 mg were measured in the temperature range of 50 °C to 270 °C at a heating rate of 10 °C/min under a nitrogen flow of 40 mL/min. An empty aluminum pan served as the reference cell.TGA was performed on dry samples weighing 5–10 mg to confirm the successful surface modification of the zeolites and to evaluate the thermal behavior of the beads. The analysis was conducted in the range 50–800 °C with a heating rate of 10 °C/min. A nitrogen atmosphere was used up to 550 °C, after which the nitrogen flow was replaced by oxygen to complete combustion of the organic compounds. Three replicates were run for each type of sample. The STARe software provided with the instrument was used for curve analysis.TM Summit/EverestTM Spectrometer  equipped with a zinc selenide attenuated total reflectance (ATR) accessory. IR spectra were acquired in the region of 4000–500 cm−1, with a resolution of 4 cm−1, and each spectrum was acquired by averaging 32 scans. To account for the background interference from the surrounding air, the data were corrected by subtracting an air background spectrum.Fourier transform infrared (FTIR) spectra of beads were recorded using a NicoletTo determine the swelling ratio and water content, the gel beads were first completely hydrated in deionized water and then dried at 50 °C for 18 h. The swelling ratio and water content were calculated using the following equations :(2)Swellswollen is the weight of the gel in the equilibrium swelling state, and Wdry is the weight of the fully dried gel. The test was repeated on 5 samples for each bead type and the results averaged. WSwelling kinetics and degradation tests were conducted to evaluate the pH response of the beads and the stabilizing effect of chitosan coating. Tests were performed on 3 mg of dried beads (18 h at 50 °C) immersed in 4 mL of solution. A preliminary test of swelling kinetics was conducted on all types of gel beads in PBS solution (pH = 7.4) for 6 h. The swelling kinetics were studied for Alg/Z5A/Cur beads with and without chitosan coating in simulated gastric fluid , acid tumoral environment (pH = 5.6), simulated intestinal fluid  and simulated colon fluid  for 6 h at 37 °C. Degradation tests were performed at different pH values in PBS solution, monitoring the beads’ behavior after 6 h.


The high vascularization of glioma highlights the potential value of anti-angiogenic therapeutics for glioma treatment. Previously, we designed a novel vascular-targeting and blood–brain barrier (BBB)-penetrating peptide, TAT-AT7, by attaching the cell-penetrating peptide TAT to a vascular-targeting peptide AT7, and we demonstrated that TAT-AT7 could target binding to the vascular endothelial growth factor receptor 2 (VEGFR-2) and Neuropilin-1 (NRP-1), which are both highly expressed in endothelial cells. TAT-AT7 has been proven to be a good targeting peptide which could effectively deliver the secretory endostatin gene to treat glioma via the TAT-AT7-modified polyethyleneimine (PEI) nanocomplex. In the current study, we further explored the molecular binding mechanisms of TAT-AT7 to VEGFR-2 and NRP-1 and its anti-glioma effects. Accordingly, TAT-AT7 was proven to competitively bind to VEGFR-2 and NRP-1 and prevent VEGF-A165 binding to the receptors by the surface plasmon resonance (SPR) assay. TAT-AT7 inhibited endothelial cells’ proliferation, migration, invasion, and tubule formation, as well as promoted endothelial cells’ apoptosis in vitro. Further research revealed that TAT-AT7 inhibited the phosphorylation of VEGFR-2 and its downstream PLC-γ, ERK1/2, SRC, AKT, and FAK kinases. Additionally, TAT-AT7 significantly inhibited angiogenesis of zebrafish embryo. Moreover, TAT-AT7 had a better penetrating ability and could penetrate the BBB into glioma tissue and target glioma neovascularization in an orthotopic U87-glioma-bearing nude mice model, and exhibited the effect of inhibiting glioma growth and angiogenesis. Taken together, the binding and function mechanisms of TAT-AT7 were firstly revealed, and TAT-AT7 was proven to be an effective and promising peptide for the development of anti-angiogenic drugs for targeted treatment of glioma. Glioma is the most common and aggressive primary brain tumor, characterized by high invasiveness and poor prognosis . Due to Hypoxia in glioma tissue stimulates the expression of the transcription factor, HIF-1α, which triggers the production of excess quantities of pro-angiogenic growth factors. One of the most pivotal pro-angiogenic growth factors involved in glioma angiogenesis is vascular endothelial growth factor A (VEGF-A) . The regMost peptides and proteins are usually functional and efficacious signaling molecules that naturally exist or are synthesized by artificial methods . CompareIntracranial delivery of therapeutic agents is primarily hindered by the BBB . The intHence, in this study, a series of in vitro and in vivo experiments were performed to explore the binding mechanism, properties, and functions of TAT-AT7. Specifically, the effect of TAT-AT7 in competition with VEGF-A165 binding to VEGFR-2 or NRP-1 was evaluated by the SPR assay. The anti-angiogenesis activities of TAT-AT7 in vitro were evaluated by the cell proliferation assay, wound healing assay, transwell invasion assay, tubule formation assay, and the Hoechst and Annexin V-FITC/PI staining assay. Western blotting was used to investigate the molecular mechanism at the cellular level. The in vivo anti-angiogenic activity of TAT-AT7 was firstly evaluated in a zebrafish model. Finally, the glioma vascular-targeting and anti-glioma effects of TAT-AT7 in vivo were evaluated in an intracranial U87-glioma-bearing nude mice model.AT7, TAT, the mixture of TAT and AT7 (TAT+AT7), and TAT-AT7 groups were set up in our following experiments. The purpose of setting up the TAT+AT7 group was to confirm that the anti-angiogenic activity of TAT-AT7 was not affected by the physical superposition of the TAT and AT7. The anti-proliferative effect was evaluated by the MTT assay. As shown in The effect of TAT-AT7 on the migration and invasion ability of HUVECs was investigated by the wound healing assay and the transwell invasion assay, respectively. As shown in In order to investigate the effect of TAT-AT7 on the formation of the tubular structure of HUVECs, Matrigel was used to simulate the structure of the basement membrane in the tube formation assay. As shown in Hoechst 33,258 staining and Annexin V-FITC/PI staining were used to investigate the effect of TAT-AT7 on apoptosis of HUVECs. Apoptotic cells could be stained to a brighter fluorescence by blue Hoechst 33,258 due to their condensed chromatin. Fluorescent images A showed The anti-angiogenesis effects of TAT-AT7 on HUVECs was demonstrated by the above experiments. In our previous study, we found that TAT-AT7 had high affinity to VEGFR-2 and NRP-1. Due to the lack of consensus signaling domains, NRP-1 mainly serves as a co-receptor for VEGF-A165- and enhanced VEGF-A165-induced VEGFR-2 downstream signal transduction, which promote angiogenesis . TherefoTo determine the effect of TAT-AT7 in competition with VEGF-A165 binding to VEGFR-2 or NRP-1, the SPR assay was conducted. The whole operation was conducted on a Biacore S200 instrument. The recombinant VEGFR-2 or NRP-1 proteins were firstly immobilized on a CM5 chip by an amine-coupling reaction. VEGF-A165 ligands with gradient concentrations in the absence or presence of different peptides were then flowed over the CM5 chip. The binding of VEGF-A165 ligands to the immobilized recombinant VEGFR-2 or NRP-1 proteins led to an increase of the refractive index on the surface of the chip, which could be converted into the fluctuation of electrical signals in the response (RU). As the concentration of VEGF-A165 was higher, the response value was also higher. However, with the addition of different peptides, the response value decreased to different degrees A.As shown in Considering the inhibitory effect of VEGF-A165 binding to VEGFR-2 and NRP-1 receptors by TAT-AT7 in the SPR analysis, we speculated that the anti-angiogenic mechanism of TAT-AT7 might be caused by the effects on the downstream signaling pathway of VEGFR-2. Next, we investigated the phosphorylation of VEGFR-2 and some key downstream kinases of VEGFR-2 to explore the molecular mechanism of anti-angiogenesis of TAT-AT7. As shown in Whether TAT-AT7 can cross the BBB is a key to treating glioma. Brain capillary endothelial cells are an essential component of the BBB; hence, the cellular uptake assay in bEnd3 cells  was conducted to evaluate the penetrating ability of TAT-AT7. The bEnd3 cells were incubated with FITC-AT7, FITC-TAT, FITC-TAT + FITC-AT7, and FITC-TAT-AT7 for 1 h, and then the cellular uptake assay was investigated by fluorescence imaging and flow cytometry. As shown in The effect of different peptides on the angiogenesis of zebrafish embryos was investigated by observing the green fluorescence intensity of the intersegmental vessel of embryos under a fluorescence microscope. As shown in The hemolysis assay was used to investigate the safety of TAT-AT7 in vivo. As shown in To investigate the glioma tissue distribution and vascular-targeting of TAT-AT7, we conducted an immunohistochemical analysis of blood vessel marker CD31-staining in orthotopic U87-mCherry-luc glioma-bearing nude mice. The anti-glioma effect of TAT-AT7 in vivo was evaluated in an intracranial U87-mCherry-luc glioma-bearing nude mice model. The luciferase in U87-mCherry-luc cells can react with the D-Luciferin potassium salt and generate bioluminescence, which can be monitored by the IVIS spectrum imaging system. The bioluminescence intensity is proportional to glioma growth. U87-mCherry-luc glioma-bearing nude mice were randomly divided into five groups. The control group was treated with normal saline.It was clear that TAT-AT7 significantly suppressed glioma growth on days 11 and 14 compared with the other groups treated with saline, AT7, TAT, and TAT+AT7 A,B. In cIn most research, AT7 or TAT are usually used as targeting or penetrating ligands to modify drug delivery systems for the treatment of different diseases ,31,32,33The BBB prevents most drugs from reaching the brain . Thus, eDifferent from traditional functional applications of TAT that are mainly used as a drug delivery tool, our present study found that TAT-AT7 presented more potent BBB penetration and anti-angiogenic effects after conjugating TAT to AT7. Then, we further performed a preliminary exploration about the potential mechanism of action mediated by TAT-AT7. Through the SPR assay, we first provided evidence that TAT-AT7 potently inhibited the VEGF-A165 binding to VEGFR-2 and NRP-1, simultaneously. A previous study found that the four C-terminal residues “LPPR” are essential for the inhibitory effect of AT7 on VEGF-A165 binding to NRP-1 . ZacharyUpon VEGF-A165 binding, the dimerization of VEGFR-2 results in the phosphorylation of specific tyrosine residues within its cytoplasmic tail. NRP-1 functions as a co-receptor to form a VEGF-A165-dependent complex with VEGFR-2 and amplifies the activity of a series of downstream transduction signaling pathways of VEGFR-2, including PLC-γ, ERK1/2, SRC, AKT, FAK, etc. ,39,40,41®, Life Technologies . Matrigel was purchased from BD Biosciences . The 96-well and 6-well plates were bought from Corning Inc. . All other reagents were of the highest commercial grade available.RKKRRQRRR (TAT), ATWLPPR (AT7), and RKKRRQRRRCATWLPPR (TAT-AT7) were synthesized from China Peptides Co., Ltd. . The Hoechst 33,258 staining kit, Annexin V-FITC/PI apoptosis detection kit, and BCA protein assay kit were obtained from Beyotime Biotechnology . The total protein extraction kit was from BestBio . The CM5 sensor chip was obtained from GE Healthcare Life Sciences . Purified recombinant human VEGFR-2 and NRP-1 receptor proteins were purchased from R&D Systems . Recombinant human endothelial growth factor-A165 (VEGF-A165) was obtained from PeproTech . Anti-ERK1/2, anti-p-ERK1/2, anti-p-VEGFR-2, anti-AKT, anti-p-AKT, anti-p-SRC, anti-p-FAK, anti-p38 MAPK, anti-p-p38 MAPK, anti-p-PLC-γ, and anti-GAPDH antibodies were purchased from Cell Signaling Technology . Anti-VEGFR-2, anti-FAK, and anti-SRC were purchased from Proteintech Group, Inc . F-12K medium, heparin, and endothelial cell growth supplement (ECGS) were from Macgene . DMEM medium and fetal bovine serum (FBS) were obtained from Gibco2 atmosphere at 37 °C. U87-mCherry-luc cells (human glioma cells) were obtained from Shanghai Sciencelight Biology Science and Technology Co. . Mouse brain microvascular endothelial cells (bEnd3 cells) were purchased from Procell Life Science and Technology Co., Ltd. . U87-mCherry-luc and bEnd3 cells were cultured in DMEM medium supplemented with 1% penicillin and streptomycin and 10% FBS, under a 5% CO2 atmosphere at 37 °C.Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC . The cells were cultured in F-12K medium supplemented with 10% FBS, 100 μg/mL of heparin, and 50 μg/mL of ECGS under a 5% COFemale nude mice (4–6 weeks old) were purchased from the Jinan Pengyue Experimental Animal Breeding Co., Ltd . Mice were raised under specific pathogen-free (SPF) conditions. All animal procedures were approved by the Ethics Committee of Shandong University and performed in accordance with the Shandong Council on Animal Care.SPR analysis is an optical technology to be used for studying ligand–receptor interactions. The SPR assay was performed using the Biacore S200 instrument . Firstly, the CM5 sensor chip was activated with EDC/NHS. Then, the recombinant VEGFR-2 and NRP-1 proteins were immobilized on the surface of the sensor chip according to the operating instructions of amine coupling. VEGF-A165 was diluted to 8, 4, 2, 1, and 0.5 nmol/L in different concentrations of peptide solution dissolved by HBS-EP buffer . The Kinetics/Affinity program was selected, and the automatic continuous injection program was set. The injection flow rate was 30 μL/min, the injection time was 120 s, and the dissociation time was 220 s. At the same time, zero-concentration injection wells and 0.5 nmol/L repeat-concentration injection wells of VEGF-A165 were set. At the end of each injection, the channels were flushed with glycine-HCl solution for 30 s until response values were returned to baseline.5 cells/well. When 80% confluence was reached, the cells were treated with 20 μM of FITC-AT7, FITC-TAT, FITC-AT7 and FITC-TAT (FITC-AT7 + FITC-TAT), and FITC-TAT-AT7 diluted in DMEM medium for 1 h . The green fluorescence images of bEnd3 cells were visualized under an inverted fluorescence microscope and the fluorescence intensity was determined by flow cytometry.In order to explore whether the permeability of TAT-AT7 to bEnd3 cells could be enhanced, a cellular uptake experiment was conducted. The bEnd3 cells were seeded into 6-well culture plates at 2 × 103 cells/well) and allowed to attach overnight. The cells were treated with AT7, TAT, the mixture of TAT and AT7 (TAT+AT7), and TAT-AT7 at a series of concentrations  in the presence of VEGF-A165 (30 ng/mL) for 48 h. Then, 20 µL of MTT solution was added to each well and further cultured for 4 h under dark conditions. After removing the medium, 150 µL of DMSO was added to dissolve formazan crystals. The 96-well plates were shaken at a low speed for 10 min. The absorbance was measured at 490 nm using a Microplate Reader .Briefly, HUVECs were seeded into 96-well plates (5 × 105 cells/well). Following overnight incubation, the monolayer HUVECs were scraped by pipette tips of 200 μL and washed by cold PBS. AT7, TAT, TAT+AT7, and TAT-AT7 (160 μmol/L) in the presence of VEGF-A165 (30 ng/mL) were added to the plates. The cells were incubated for another 48 h. Images were captured at 0 h, 24 h, and 48 h using an inverted microscope. The cell migration rate was counted by the ImageJ software.HUVECs were firstly seeded into 6-well culture plates (3 × 104 cells/well. F-12K medium (containing 20% serum) was added to the lower chamber. HUVECs were incubated with AT7, TAT, TAT+AT7, and TAT-AT7 (160 μmol/L) in the presence of VEGF-A165 (30 ng/mL) in F-12K medium supplemented with 10% FBS and were incubated at 37 °C for 24 h. Then, the medium was aspirated, and the chambers were washed with PBS and placed in a fixative for 30 min, stained with 0.1% crystal violet for 30 min, washed with PBS, and dried. An inverted fluorescence microscope was used to photograph the chambers, and the number of invaded cells was counted. The cells that migrated to the bottom of the chamber were photographed with an inverted microscope.Matrigel was diluted in serum-free F-12K medium (dilution ratio 1:5) and was coated onto each transwell chamber and incubated at 37 °C for 2 h. HUVECs were seeded in the upper compartment of the transwell chamber at a density of 2 × 104 cells/well) and incubated with AT7, TAT, TAT+AT7, and TAT-AT7 (160 μmol/L) in the presence of VEGF-A165 (30 ng/mL) for 6 h. The tubular structures were observed under an inverted microscope and the total branching lengths were calculated by the ImageJ V 1.8.0 software.Matrigel was added to 96-well plates with 60 min of incubation at 37 °C for solidification. HUVECs were seeded into the Matrigel-coated plates (3 × 105 cells/well). After overnight incubation, the cells were washed with PBS and treated with AT7, TAT, TAT+AT7, and TAT-AT7 (320 μmol/L) for 24 h. After discarding the supernatant and washing with cold PBS, the cells were fixed in 4% paraformaldehyde and stained with Hoechst 33,258 for 10 min. Then, the cells were washed with cold PBS and observed using an inverted fluorescence microscope.HUVECs were seeded into 6-well culture plates (3 × 10HUVECs were treated with different peptides (the methods were the same as the Hoechst staining). After 24 h, cells were collected by trypsin digestion and washed with PBS. Annexin V-FITC/PI staining was conducted according to the manufacturer’s instructions for the Apoptosis Detection Kit. Briefly, 195 μL of Annexin V-FITC/PI dilution buffer was added to resuspend the cells. Then, 5 μL of Annexin V-FITC and 10 μL of PI staining solutions were added and gently mixed with the cells. After incubation at room temperature in the dark for 10 min, the apoptotic cells were detected by flow cytometry.5 cells/well and incubated overnight. After washing with PBS, cells were treated with AT7, TAT, TAT+AT7, and TAT-AT7 (320 μmol/L) in the presence of VEGF-A165 (30 ng/mL) for 90 min. Two control groups (F-12K medium in the absence or presence of VEGF-A165) were set. The cells were washed with cold PBS and lysed with lysis buffer (protease and phosphatase inhibitors) on the ice for 30 min, and then centrifuged at 4 °C for 15 min at 14,000 r/min. The supernatant was collected, and its total protein concentration was determined by the BCA protein assay kit. Equal amounts of proteins were separated by 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with specific antibodies at 4 °C overnight. Then, the membranes were washed with TBST  and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The proteins on the membranes were visualized with enhanced chemiluminescence agents.For Western blotting analysis, HUVECs were seeded into 6-well culture plates at 3 × 10Female and male transgenic zebrafish were reared in alternating environments of 14 h light and 10 h dark, respectively. After a certain period, healthy, sexually mature female and male zebrafish were mated in a 1:1 ratio. After 24 h, the fertilized eggs were removed, disinfected, and washed, and then placed in embryo culture water and cultured in an incubator at 28 °C for 24 h. The fertilized eggs were washed with chain enzyme protease E solution (1 mg/mL) to remove the egg membrane on the surface and placed under a microscope to observe and select the normally developing zebrafish embryos, and then placed into a 24-well plate. AT7, TAT, TAT+AT7, and TAT-AT7 at a concentration of 25 µmol/L were added to the plates, respectively. The control group was added with the same volume of embryo culture water, and then placed in an incubator at 28 °C for 24 h. Zebrafish embryos were randomly removed from each group and observed under a fluorescence microscope. The total length of zebrafish internode vessels was calculated and counted.The fresh blood of mice was centrifuged at 2000 r/min for 10 min, and the upper layer of serum was discarded, then mixed with normal saline and centrifuged at 2000 r/min for 10 min, repeated three times, and the supernatant was removed to obtain the lower layer of red blood cells. Then, 1 mL of red blood cells was diluted to 50 mL with normal saline. AT7, TAT, TAT+AT7, and TAT-AT7 solutions diluted with normal saline  were added to equal volumes of the red blood cell suspension, respectively. The samples were incubated at 37 °C for 1 h, centrifuged at 2000 r/min for 10 min, and photographed. Light absorption of samples was measured at 540 nm on a microplate reader.Cell hemolysis rate (%) = /(OD positive control group − negative control group) × 100%.n = 3). FITC-AT7, FITC-TAT, and FITC-TAT-AT7 (30 mg/Kg) were injected into mice via the tail vein on day 8. The control group was only injected with normal saline. After 1 h, the mice were sacrificed, and the brains were collected and fixed with 4% paraformaldehyde for 24 h. Then, the fixed brains were dehydrated by 15% and 30% sucrose, successively, frozen with O.C.T. embedding reagent, and then cut into frozen brain tissue slices. Then, sections of slices were incubated with rabbit anti-murine CD31 antibody, followed by treatment with Cy5.5-labeled goat anti-rabbit IgG secondary antibody. The slices were further stained with DAPI for 5 min and observed using a confocal microscope.The orthotopic U87-mCherry-luc glioma-bearing nude mice model was firstly established. Female nude mice were anesthetized by isoflurane and fixed on a ventricle stereotaxic instrument. U87-mCherry-luc cells were collected after trypsin digestion and then injected into the right striatum of the brain of nude mice. On the 4th day after the surgery, the bioluminescence of the glioma tissue was detected by the IVIS kinetic imaging system . The mice were randomly divided into four groups according to the bioluminescence intensity (n = 5) according to the bioluminescence intensity of glioma measured on day 4. The mice in five of the groups were intravenously injected with normal saline, TAT, AT7, TAT+AT7, and TAT-AT7 (20 mg/kg/day), respectively, every day for 11 days. The bioluminescence intensity was measured on days 4, 7, 11, and 14, respectively. Nude mice were weighed daily. On day 15, the mice were sacrificed, and the glioma-bearing brains were harvested. The brain tissues were fixed with 4.0% paraformaldehyde, embedded in paraffin, and cut into slices. The TUNEL staining and CD31 immunohistochemical staining were conducted according to standard histological procedures.The orthotopic U87-mCherry-luc glioma-bearing nude mice were randomly assigned to 6 groups (t test or one-way ANOVA (GraphPad Prism 6.0). The value of p < 0.05 was considered as statistically significant.The data were expressed as mean ± standard deviation (SD). All statistical analyses were determined by Student’s In conclusion, we demonstrated that the chimeric peptide TAT-AT7 had a targeted binding ability to VEGFR-2 and NRP-1. TAT-AT7 could inhibit angiogenesis by downregulating phosphorylation of VEGFR2-related signaling pathways. Moreover, TAT-AT7 showed a strong penetrating ability into glioma tissue and a glioma neovascularization-targeting property. In vivo experiments showed that TAT-AT7 could effectively inhibit glioma growth and angiogenesis. In conclusion, TAT-AT7 may present a new strategy for targeted therapy of glioma.


In 2010, Madariaga virus (MADV) newly emerged in Darien, Panama, where Venezuelan equine encephalitis virus (VEEV) is endemic. We noted an inverse relationship between VEEV and MADV seroprevalence in humans, prompting the study of heterologous immunity in alphaviral infection.Using patient samples from Panama, we examined antibody-dependent cell mediated cytotoxicity (ADCC) and antibody-dependent cell mediated lysis assays (ADCL) to three viruses: Mayaro virus (MAYV), Madariaga virus (MADV), and the vaccine strain of Venezuelan equine encephalitis virus (TC83). We developed an ADCC assay using adherent target cells , and high-throughput immunofluorescence microscopy. We determined ADCL activity using alphavirus-infected K562 cells and flow cytometry. We further defined the specificity of antibodies by plaque reduction neutralization tests, whole viral antigen and virus-specific recombinant glycoprotein (rGP) ELISAs.Sera from alphavirus-exposed individuals demonstrated significantly greater ADCC and ADCL activity compared to alphavirus-naïve sera. Blocking CD16 abrogated this effect, confirming the role of natural killer cells in inducing ADCC and ADCL. The relationship is asymmetric; prior VEEV exposure yields a high degree of homologous and heterologous ADCC and ADCL activity, whereas MADV and MAYV exposure results in heterologous ADCC and ADCL activity no different than alphavirus-negative controls. We observed a strong association between ADCC/ADCL activity and whole viral antigen ELISA titers, but not rGP ELISA nor neutralization titers, suggests that the viral antigens that elicit ADCC and ADCL may be nonstructural proteins.in vitro by ADCC and ADCL, but not by neutralization. This has implications for vaccine development. Eliciting an antibody response that is not only neutralizing, but also generates antibodies with ADCC and ADCL activity may produce broader protection against viral variants or antigenically similar viruses. Additionally, these results provide insight into immunological mechanisms underlying the emergence potential of a novel virus.Here we demonstrate that VEEV antibodies are active against heterologous alphaviral infections All Authors: No reported disclosures


The expected economic burden on the healthcare system because of quarantining patients with monkeypox virusAlshahrani determines the expected economic cost for healthcare systems due to quarantine in the case of the monkeypox virus. A systematic literature review of studies on similar virus outbreaks was performed. The findings affirm that quarantine effectively mitigates the spread of a virus outbreak, but it has high direct and indirect costs that can only be justifiable for a dangerous virus with high mortality. The monkeypox virus presents moderate risk, unlike high-risk diseases for which quarantine is mandatory. The study recommends the introduction of mass vaccination programs and public awareness and sensitization forums to inform the population about the best behavioral practices to curb the spread of monkeypox virus.see page 231 A retrospective study in a city in the Southeastern region of TurkeyCOVID-19 infection during pregnancy. Akgül et al evaluate maternal and fetal clinical outcomes in SARS-CoV-2 infected pregnant women during the whole period of the pandemic in a single province in the southeast of Turkey. They concluded that in the 3rd trimester of pregnancy, obesity and hypothyroidism were found to be risk factors for severe-critical cases of COVID-19 infection, and the clinical course was more severe with a higher rate of mortality in the recent period of the pandemic.see page 268A cross-sectional study in Saudi populationSelf-care practices among heart failure patients. Aljohani describes self-care practices among Saudi heart failure (HF) patients and identify sociodemographic characteristics contributing to self-care practices. He concluded that Self-care practices in this study showed higher scores than those reported in international studies. Further studies are warranted to explore everyday self-care needs and challenges among HF patients.see page 277Patellar height changes after treatment of tibia plateau fractures. A radiological analysisDoğan et al determine the impact of tibial plateau fractures on patellar height and the factors affecting this impact in in a retrospective prognostic study. They concluded that Long-term functions of tibial plateau fractures should be evaluated not only with a painless range of motion but also with patellar height. It should be noted that the Luo classification, which evaluates the plateau 3-dimensionally, may be associated with changes in postoperative patellar height valuessee page 306


Dermacentor silvarum ticks near the China–North Korea border. Phylogenetic analysis showed 71.9%–73.0% nucleic acid identity to the recently discovered Songling orthonairovirus, which causes febrile illness in humans. We recommend enhanced surveillance for infection by this new virus among humans and livestock.We isolated a new orthonairovirus from  Orthonairovirus, family Nairoviridae, include the consequential tick-transmitted pathogens Crimean–Congo hemorrhagic fever virus and Nairobi sheep disease virus, as well as other poorly characterized viruses that have been found in ticks and mammals. Orthonairovirus virions are spherical in shape (80–120-nm diameter) with 3 single-stranded RNA segments 17.1–22.8 kilobases in length and a membrane envelope , a district of the city of Yanbian in eastern Jilin Province, China, near the border with North Korea. We identified captured ticks according to morphologic keys and stored them at 4°C with wet cotton. We collected 264 ticks of 3 species—29 https://www.qiagen.com) and inoculated supernatants onto a monolayer of African green monkey kidney (Vero) E6 cells. After 3 successive passages, we observed cells for cytopathic effects. The inoculate from Dermacentor silvarum ticks, designated as YB_tick_2021_24, caused cytopathic effects in Vero E6 cells 96 h after inoculation , and performed 150 bp paired-end sequencing using the Illumina MiniSeq System. We filtered reads on the basis of their length and mean quality values. We prepared contigs by de novo assembly and subjected them to BLASTx alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi) at E value <10−4 against the nonredundant protein and viral proteome databases of the National Center for Biotechnology Information. We used Bowtie 2 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml) to remap the clean reads to the generated virus-related contigs (https://www.ncbi.nlm.nih.gov/orffinder) and calculated sequence similarities using BLAST. We extracted viral RNA from infected culture supernatants using a QIAGEN QIAamp Viral RNA Mini Kit, synthesized cDNA, prepared DNA libraries using an Illumina Nextera XT Kit  (https://www.megasoftware.net) by using the neighbor-joining method with a bootstrap test for 1,000 replicates , which produced 266 virus-related contigs. Three contigs, the 1,516 bp small (S), 3,936 bp medium (M), and 12,133 bp large (L) segments, were annotated to Songling virus (SLV), a previously reported orthonairovirus . Phylogenetic analysis showed the strain belongs to the genus d to SLV  . ThoDermacentor silvarum ticks collected in China near the China–North Korea border. Nucleotide and amino acid sequence homologies, combined with phylogenetic analysis of other orthonairovirus genomes, suggested that Antu virus is a new member of the genus Orthonairovirus, genetically related to SLV. Tamdy virus and SLV are orthonairoviruses reportedly able to infect human and livestock (We identified a novel orthonairovirus, Antu virus, in Dermacentor silvarum ticks near the China–North Korea border. Additional information on a new orthonairovirus species isolated from


Purpose: The aim of this study was to evaluate the influence of different SARS-CoV-2 strains on the functional capacity of athletes. Methods: In total, 220 athletes underwent cardiopulmonary exercise testing (CPET) after coronavirus infection and before returning to sports activities. Eighty-eight athletes were infected by the Wuhan virus, and 66 were infected during the Delta and Omicron strain periods of the pandemic. Results: The CPET results showed significantly decreased maximal oxygen consumption, ventilatory efficiency, and oxygen pulse in athletes who were infected with Wuhan and Delta strains compared to athletes who suffered from Omicron virus infection. An early transition from aerobic to anaerobic metabolic pathways for energy production was observed in the Wuhan and Delta groups but not in athletes who were infected with the Omicron strain. There were no differences in the obtained results when Wuhan and Delta virus variants were compared. Conclusion: These results suggest that the Wuhan and Delta virus strains had a significantly greater negative impact on the functional abilities of athletes compared to the Omicron virus variant, especially in terms of aerobic capacity and cardiorespiratory function.  The pandemic of the SARS-CoV-2 virus has left enormous consequences for the lives and health of ordinary people, as well as athletes . The virAt first, it was assumed that older people and those with underlying medical conditions were more likely to become infected and develop serious illnesses. Considering that athletes belong to a young and healthy population, at the beginning of the pandemic, the recommendations for returning to sports activities included only a physical examination and electrocardiogram (ECG) in rest, with basic laboratory analyses, following a minimum of fourteen days of home isolation ,7. Over Since the beginning of the pandemic in March 2020, the SARS-CoV-2 virus has mutated over time, resulting in genetic variations in the population of circulating viral strains. These mutations may impact virus transmission or the severity of symptoms in infected individuals . AccordiTwo hundred and twenty (N = 220) elite athletes from Serbia participated in this prospective cohort study. In total, 112 soccer players (age 23.05 + 4.64) from five professional senior Serbian teams  and 108 basketball players (age 24.52 + 4.80) from six professional Serbian teams (Euroleague and ABA League) were involved in the study. This research was conducted during the period from September 2020 to September 2022. The criteria for inclusion in the study were prior SARS-CoV-2 infection, which had been confirmed by a polymerase chain reaction test. There were two indications for conducting the PCR tests. The first referred to athletes who had clear symptoms of infection. Another indication for conducting the PCR test was the mandatory testing of athletes before every official match, according to the proposals of domestic and European competitions. The Genome Sequencing Center within the Institute of Molecular Genetics and Genetic Engineering of Serbia conducted sequencing of the coronavirus genome to identify new coronavirus strains. Genome sequencing was carried out when a new strain was suspected and continued until the point when it was evident that the dominance of a new variant of the virus had emerged. Accordingly, study subjects were divided into three groups depending on the period of the pandemic during which they had been infected by SARS-CoV-2 and the dominance of one of the three strains of the virus in that time interval .n = 88). Genome sequencing at the beginning of this period indicated the dominance of the Wuhan strain. The second group of athletes were subjects who were thought to have been predominantly infected with the Delta strain (n = 66). These participants had PCR positive tests in the period from August 2021 to January 2022. The third research group consisted of athletes tested from February 2022 to September 2022 (n = 66). By sequencing the genome of the virus during this period of the pandemic, it was concluded that the Omicron strain was dominant. The first group of participants consisted of athletes who were infected with the SARS-CoV-2 virus in the period between September 2020 and July 2021 . After the cessation of symptoms of SARS-CoV-2 infection and/or a negative control PCR test, study participants underwent medical examinations with the aim of deciding on their capacity to return to sports activities. The medical examination included an electrocardiographic examination at rest, blood pressure measurement, and auscultation of the heart and lungs . Additionally, laboratory analyses were performed with the aim of evaluating inflammatory and cardiac biomarkers, such as C-reactive protein (CRP), D-Dimer, NT-proB-type Natriuretic Peptide (nt-pro BNP), and high-sensitivity cardiac troponin T (hs-cTnT). Along with laboratory analyses, transthoracic 2D echocardiography was done to ensure that there was no ongoing acute inflammatory process and/or underlaying myo/pericarditis as absolute contraindications for performing cardiopulmonary exercise testing (CPET), which was the final medical exam prior to the decision of whether or not to return to play. 2/min), and a respiratory exchange ratio greater than 1.10, together with reached volitional exhaustion. All tests were performed by medical doctors, and the test equipment was routinely calibrated with both volume and gas calibration before each testing procedure. CPET, as a maximal symptom-limited exercise test, was performed to evaluate the health status and functional capacity of athletes. Maximal exercise testing was performed on a treadmill. Subjects were equipped with a facemask, heart rate monitor , and portable ECG device  to perform the test. According to the protocol for professional athletes, the initial speed and inclination were set at 6 km/h and 3°, respectively. Every 40 s, the treadmill speed was increased by 1 km/h, while the inclination remained constant throughout the test. Oxygen consumption kinetics were measured continuously using a breath-by-breath analysis technique . Heart rate was monitored by a portable ECG device. A test was considered maximal if participants achieved 90% or more of predicted maximal heart rate for age and gender (220–age), a plateau in oxygen consumption despite increased workload (plateau < 150 mL O2/HR), as an indirect indicator of left ventricular function (the volume of oxygen ejected from the ventricles with each cardiac contraction), was measured and assessed by a Wasserman 9-Panel Plot . The efficiency of the ventilatory pump at various workloads was continuously evaluated using Panel 4 of the Wasserman 9-Panel Plot , as an objective and accurate indicator of cardiorespiratory fitness and aerobic endurance, was evaluated at the end of the CPET. The plateau in oxygen consumption was considered when determining the final VO2 max value  was also a sign of reaching the first threshold. Respiratory exchange ratio (RER), as a quotient of metabolic production of carbon dioxide and the uptake of oxygen (CO2/O2) was used to evaluate the point of RCP, or second ventilatory anaerobic threshold. The onset of absolute anaerobic metabolism was determined by measuring the heart rate at RER = 1. Furthermore, the simultaneous sudden increase in ventilatory equivalents for oxygen and carbon dioxide, as well as the sudden drop in the end-tidal partial pressure for carbon dioxide (PetCO2) indicated reaching the second ventilatory threshold. At this point, ventilatory requirements for delivering oxygen to the muscle cells and the removal of carbon dioxide into the external environment are extremely high. Additionally, the RER value was calculated at the end of the test  to assess the metabolic response to the maximal effort and level of achieved anaerobic exertion. The heart rate was obtained at the first ventilatory anaerobic threshold (VAT) and second ventilatory anaerobic threshold (or respiratory compensation point (RCP)) to determine the intensity of effort at which the transition from aerobic to anaerobic energy sources occurs. VAT and RCP were measured by using the “thresholds” panels of the Wasserman 9-Panel Plot  was used, as was the Post Hoc Bonferroni test for multiple comparisons. Statistical data processing was performed using the statistical package SPSS 20.0 for Windows. Differences were considered significant when the The results of laboratory analyses that were a prerequisite for conducting CPET are shown in Descriptive statistics for the obtained CPET variables are shown in 2 max values in athletes who were infected with the Omicron virus variant compared to those who had been infected by the coronavirus disease during the dominance of the Wuhan and Delta strains (p < 0.01). This result was also confirmed by the Post Hoc Bonferroni test (p < 0.01). When we compared aerobic capacity between the Wuhan and Delta groups of participants, there was no statistically significant difference. Despite this fact, 2 max than the mean calculated value and compared to the same data within the Delta group. The one-way ANOVA test showed statistically significantly higher VO2/HR values than those infected with the Wuhan and Delta strains  production was absent during CPET in the Omicron group when compared to the Wuhan and Delta strains. Achieved heart rate values at VAT and RCP were much higher in these participants compared to the athletes from the Wuhan and Delta groups. On the other hand, there was no statistically significant difference either in terms of maximal achieved heart rate at the end of CPET or in terms of the three-minute heart rate recovery among all three study groups. The heart rate analysis and the influence of different SARS-CoV-2 strains on this data, during the different phases of the maximum effort test, are shown in p < 0.01). Exposure to early and pronounced metabolic fatigue during the period of dominance of the Wuhan and Delta strains was recorded by significantly high maximum RER values at the end of the CPET tests when compared to the Omicron group of athletes. The one-way ANOVA and Post Hoc Bonferroni test confirmed a statistically significant difference between the achieved RER values at the end of the test when we compared Wuhan and Omicron and Delta and Omicron strains (p > 0.05). During CPET, no malignant disorders of rhythm, conduction, or pathological changes of the ST-T segment were observed in any of the examined athletes. All athletes received permission to return to sports activities with a gradual entry into the training process, especially in the early stages of the pandemic, given that their functional capacity was significantly compromised.There was no statistically significant difference between athletes who were infected with the Wuhan and Delta variants of SARS-CoV-2 virus when considering all the evaluated variables , there was an obvious increase in both aerobic capacity and aerobic economy as the pandemic progressed. In other words, athletes who were infected with the Omicron strain had a significantly higher VO2 max and expended oxygen more economically, delaying the early onset of anaerobic metabolism and fatigue. The results showed significantly decreased aerobic capacity of all tested athletes. Maximal oxygen consumption, as a measure of cardiorespiratory fitness, was much lower in the participants compared to the values anticipated for professional soccer and basketball players. Soccer players possess excellent endurance, with VOrformers . On the L/kg/min . In genenfection ,19,23. E2 between 20 and 30) [2 max values in these athletes. Furthermore, O2/HR values were also within normal values for professional athletes (>20 mL/beat) throughout the pandemic [Even though ventilatory efficiency was within normal values (VE/VCO and 30)  in all tpandemic , but sig2/HR were within normal limits, the coronavirus in all three groups of subjects did not impair external respiration, lung, or cardiovascular function. Therefore, low values of aerobic capacity and aerobic efficiency in all three groups of subjects, especially in athletes who suffered from Wuhan and Delta strain infection, can probably be explained by impaired internal respiration. Oxygen delivery to muscle cells was apparently adequate, but tissue utilization of oxygen was compromised. This could potentially be explained by the reduced number of mitochondria at the level of muscle cells and, thus, the inability to adequately utilize oxygen to produce ATP. The latest research related to pandemics indicates the potential of the coronavirus to damage mitochondria as the main oxygen organelles [Hypoxemia-associated changes in external respiration are the first objective indicators of the clinical signs of respiratory failure after SARS-CoV-2 infection . Given tganelles ,31. The 2 max VE/VCO2 slope, and O2/HR values, together with delayed transition from aerobic to anaerobic metabolism, were observed in the Omicron group of athletes when compared to other evaluated strains. Additionally, only a few studies have dealt with the impact of different virus strains on the relevant clinical parameters in SARS-CoV-2 patients. The results of one such study showed that the Wuhan strain had a less favorable effect on the erythropoiesis process and led to more pronounced hypoxia in SARS-CoV-2 patients compared to other variants of this virus in later phases of the pandemic . ConsideThe main results of the study indicate that the potential of the virus to impair an athlete’s functional abilities decreases as the pandemic progresses, which greatly facilitates a safe return-to-play decisions for both sports physicians and coaches. Although no pathological changes were observed after the SARS-CoV-2 infection, the virus overall reduced the functional capacity of the athletes, especially in the early stages of the pandemic during Wuhan and Delta strain dominance. This suggests that a gradual and cautious return to sports activities is still necessary to avoid exacerbation of potential underlying accute myo/pericarditis or lung disease.


Rhizoctonia solani, which has caused a rice sheath blight, has hosted many viruses with different morphologies. It causes significant crop diseases that adversely affect agriculture and the economy. Rice sheath blight threatens the 40% of the global population that relies on rice for food and nutrition. This article reviews mycovirology research on Rhizoctonia solani to demonstrate scientific advances. Mycoviruses control rice sheath blight. Hypovirulence-associated mycoviruses are needed to control R. solani since no cultivars are resistant. Mycoviruses are usually cryptic, but they can benefit the host fungus. Phytopathologists may use hypovirulent viruses as biological control agents. New tools are being developed based on host genome studies to overcome the intellectual challenge of comprehending the interactions between viruses and fungi and the practical challenge of influencing these interactions to develop biocontrol agents against significant plant pathogens.Mycoviruses, or fungal viruses, are prevalent in all significant fungal kingdoms and genera. These low-virulence viruses can be used as biocontrol agents to manage fungal diseases. These viruses are divided into 19 officially recognized families and 1 unclassified genus. Mycoviruses alter sexual reproduction, pigmentation, and development. Spores and fungal hypha spread mycoviruses. Isometric particles mostly encapsulate dsRNA mycoviruses. The widespread plant-pathogenic fungus  Poaceae family, is grown for its seeds and used as a common cereal grain in tropical, subtropical, and mild temperate climates. Almost 40% of the world’s population relies on rice for food and nutrition, making it the second most essential crop after wheat .,44.PartiR. solani contains the first dsRNA element ever reported, being discovered by Butler and Castano ..R. solanBarnaviridae [Rhizoctonia solani barnavirus 1 (RsBV1) and barnavirus 1, belonging to the genus barnavirus, have been identified [There is presently just one species and one genus of barnavirus in the family aviridae . The modentified . The polBenyviridae [Benyvirus, and the individuals of this family are linked to cell-to-cell migration [R. solani 42304-9a and R. solani AG-2.2 LP BR2 were discovered to have two different viruses, known as Rhizoctonia solani Beny-like virus 1 (RsBenV1) and Rhizoctonia solani Beny-like virus 3 (RsBenV3), which were both identified and partially sequenced. Only one region of each genome was identified as encoding a putative RdRp associated with benyviruses and beny-like viruses. RsBenV1/42304-9 shares a close resemblance to a recognized individual of the Benyviridae family, whereas RsBenV1/BR2 resembles the Sclerotium rolfsii beny-like virus 1.Positive-sense ssRNA viruses of plants with rod-shaped particles (virions) are capped and polyadenylated genomes. Their lengths range from 1.3 to 6.7 kb, and they are found in the family yviridae . Four spigration ,75. R. sBotourmiaviridae (Botoulivirus (2.9 kbp long), Magoulivirus (2.3 kbp long), Ourmiavirus , and Scleroulivirus ). Just 59% to 87% of the Rhizoctonia solani ourmia-like virus 1 (RsOLV1) genome has been described, and an essential investigation revealed similarities to the RdRps of Ourmiavirus genus members, including the Cassava virus C, Ourmia melon virus, and the Epirus cherry [Ourmiavirus genus developed by reassorting the genomic segments of viruses that infected both plants and fungi. These viruses are tripartite, with each segment coding for one protein: RdRp, putative CP, and protein movement (MP) [S. sclerotiorum and solely encoded the RdRp protein, which is sufficient for infection, replication, and transmission. Moreover, research on SsOLV4 implies that the family Botourmiaviridae needs a new genus. As a result, fungal ourmia-like viruses only have one segment at this time. Today, Rhizoctonia magoulivirus 1 (RsOLV1) is the family Botourmiaviridae, a Magoulivirus genus and legally recognized species. In addition, the evolutionary phylogenetic tree grouped RsOLV 2–5 and Agaricus bisporus virus 15 into a potentially new genus within the family Botourmiaviridae, and even shows a new relationship with the family that is named Basidionarnaviridae [Ten species and four genera make up the family s cherry ,76. It ient (MP) . Neverthent (MP) ,76. Accoaviridae . About 7aviridae .Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus are six of the family’s current genera. The tripartite linear (+) ssRNA genome of the Bromoviridae family is around 8 kb long [Cucumovirus and Ilarvirus have an extra overlapping ORF. For the individuals of the genera Cucumovirus, Anulavirus, Ilarvirus, and Bromovirus, the family Bromoviridae generates particles (virions) that are either spherical or quasi-spherical or bacilliform, like the members of the genera Alfamovirus, Ilarvirus, and Oleavirus [ kb long ,78. Putaleavirus .Alphaflexiviridae, Deltaflexiviridae, Betaflexiviridae, and Gammaflexiviridae are highlighted by the collective name “flexiviruses” which belongs to the order Tymovirales. The filamentous virions of flexiviruses possess a monopartite (+) ssRNA polyadenylated genome that is between 6.5 and 9.5 kb long and codes for a replication-associated polyprotein between 150 and 250 kDa in size [Botrytis virus F (BotV-F), which is related to the family Gammaflexiviridae and the genus Mycoflexivirus, was the first mycovirus identified in the order Tymovirales [Deltaflexivirus have been described within the family Deltaflexiviridae: Sclerotinia deltaflexivirus 1 (SsDFV1), soybean-associated deltaflexivirus 1 (SlaMFV1), and Fusarium deltaflexivirus 1 (FgDFV1) [Rhizoctonia solani flexivirus 1 (RsFV-1) is the only flexivirus that is fully sequenced and has been found to affect the pathogenic fungus R. solani [R. solani AG2- 2IV/DC17, has 10,621 nts without the poly-A tail. One protein, encoded by RsFV-1, is connected to those of other Tymovirales members [Tymoviridae is a family that includes (+) ssRNA viruses between 6.0 and 7.5 kb in size [Tymoviridae, including three genera: Marafivirus, Maculavirus, and Tymovirus [The families  in size . It is k in size . Botrytiovirales . Three s(FgDFV1) . Rhizoct. solani . The (+) members . Tymovir in size . Forty-oymovirus . Endornaviridae has RNA genomes that are unencapsidated, and their size ranges from 8.7 to 16.6 kb. Just one ORF in the family codes for a polyprotein [Endornaviruses are chronic infections of plants, fungi, and oomycetes that do not manifest symptoms in their hosts [Endornaviridae are transmitted vertically through sporulation and horizontally through anastomosis [R. solani AG-3PT strain RS002 (RsEV-RS002) was partially characterized and tentatively entitled Rhizoctonia solani endornavirus.A family of viruses known as yprotein . The polyprotein . Endornair hosts ,85. In fstomosis , while iHypoviridae [Cryphonectria parasitica initially sparked interest in them. Via hyphal anastomosis, the members of the hypovirus can spread to virulent isolates horizontally [R. solani hypovirus genome has not yet been found; nevertheless, the NGS technique was previously used to identify whole ORFs of Rhizoctonia solani hypovirus 1 (RsHV1) and partial ORFs of Rhizoctonia solani hypoviruses 2 and 3  [Hypoviridae, a new genus, Megahypovirus, was also suggested to fit the huge genomes of RsHV1 and SsHV2, as well as RsHV2, RsHV3, and Agaricus bisporus virus 2 [Fusariviridae. Rhizoctonia solani fusarivirus 1, 2, and 3  were described [One identified species, Hypovirus, and four others with capsidless, mono-segmented (+) ssRNA genomes with a size ranging from 12.7 to 9.2 Kbp make up the family oviridae . One or oviridae . Additiooviridae ,90. The ctively) . With tw virus 2 . Also, tescribed . The 11 escribed .Narnaviridae and Mitoviridae families. Their linear (+) ssRNA genomes, which range in size from 1.7 to 3.6 kb, include a single ORF that encodes a putative RdRp and lacks a capsid. Mitoviridae and Narnaviridae each have one genus, Mitovirus (five species) and Narnavirus (two species), respectively [Narnavirus has also been discovered [C. parasitica, many mitoviruses have been found in phytopathogenic fungi [R. solani does not include any narnaviruses or complete mitovirus genome sequences. Forty incomplete mitovirus genome sequences associated with R. solani have been identified [The simplest known viruses are those belonging to the ectively . Accordiectively , all knoscovered . Althougscovered ,94. In cic fungi . The patentified ,65,95.Togaviridae family, which includes 31 species in total [Togaviridae family and range in size from 10 to 2 Kbp [R. solani is not infected by any members of the genus Alphavirus. However, recently, partial genomic sequences belonging to the Togaviridae family, including Rhizoctonia solani alphavirus-like 1, 2, and 3, were found in R. solani AG-2 LPin  [The Eastern equine encephalitis virus (EEEV), the Western equine encephalitis virus (WEEV), the Venezuelan equine encephalitis virus (VEEV), the Sindbis virus (SINV), the Ross River virus (RRV), the Semliki Forest virus (SFV), and the Chikun virus (SFV) are all members of the in total . Arbovirin total . Small eto 2 Kbp . The virto 2 Kbp . To the LV3/BR8) . The RsALV3/BR8) .Aspiviridae, formerly known as Ophioviridae, is a family of adaptable filamentous viruses that belong to the order Serpentovirales and are known to infect plants [Aspiviridae has seven species, including the genus Ophiovirus. The (−) ssRNA genomes of the family Aspirividae range in size from 11.3 to 12.5 kb and are divided into three to seven segments [Rhizoctonia solani negative-stranded RNA virus 1-3 (RsNSRV1-3) is a recently identified ophiovirus-related incomplete viral genome sequence that infects soil-borne R. solani strains. The lettuce ring necrosis ophiovirus and other members of the Aspiviridae family have RNA1s that encode big ORFs with considerable similarities to the L-protein domain. The Fusarium poae negative-stranded RNA virus1 (FpNSV1), isolated from the pathogenic fungus Fusarium poae, has also been proposed as a new family, Betamycoserpentoviridae, in the order Serpentovirales [t plants . Currentsegments . Rhizoctovirales .Holothuria polii [The Arenaviridae, Cruliviridae, Hantaviridae, Fimoviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Phasmaviridae, Peribunyaviridae, Phenuiviridae, Tospoviridae, and Wupedeviridae families are considered herein. Many (ssRNA) fungal viruses linked to bi- and tri-segmented (ssRNA) viruses, like phenuiviruses and peribunyaviruses, were found in plant-pathogenic fungi after metatranscriptomics analysis . For insia polii .Cryphonectria hypovirus 1 (CHV1), which was successfully used in Europe to eradicate the plant disease Cryphonectria parasitica, the cause of chestnut blight [Mycovirus infections are frequently asymptomatic (cryptic), although research focuses on possible hypovirulence, a feature that might emerge in the setting of long-term biological control of fungi. The best example is t blight . The tert blight . The “Lat blight . To studt blight .R. solani’s wide host range and soil-borne and saprotrophic nature, attempts to manage rice sheath blight using agronomic measures, such as breeding techniques, chemical fungicides, and crop rotation, have been ineffectual. The pathogenic fungus R. solani showed tenacity even when large amounts of chemicals, such as fungicides, were used because of significant crop losses. The fungus R. solani is notable for not producing conidia , so its capacity to be widely distributed in soil is limited. Another review on these viruses is being carried out to identify pathogenicity-related hypovirulence from R. solani while supplying novel viral resources and techniques for the biological control of R. solani-caused illness. The review will present advanced knowledge of fungal virus varieties, evolution, interaction with hosts, and the application of fungal viruses in managing sheath blight disease. This review will advance our understanding of the evolution of the many mycoviruses. Also, this review will increase our understanding of new mycoviruses and their potential application as biological control agents against plant-pathogenic fungi.One of the most significant fungal diseases of rice in the world is rice sheath blight. One of the primary issues limiting the growth of ratoon rice is the risk of the occurrence of rice sheath blight, which prevents the axillary bud germination of ratoon rice. Due to O. sativa) from R. solani while minimizing the use of chemical fungicides, new options must be developed, preferably with minimal environmental effects. Using fungal viruses to manage agricultural diseases has advanced recently. Moreover, it is known that some viruses, some of which are currently unidentified, are hosted by the pathogenic fungus R. solani. Mycoviruses from R. solani primarily linked to hypovirulence or potentially acting as biocontrol agents can be screened to learn new information and develop a new preventative technology system for managing R. solani.To safeguard crops like rice (


The increased number of carboxyl groups in the bead matrix improved the removal efficiency of the adsorbent without compromising the water throughput rate; being as high as 17 000 L h−1 m−2 bar−1. The absorptivity of the beads was evaluated with UV-vis for the removal of the dye Methylene Blue  from spiked water and energy dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) elemental analyses for the removal of Cd2+ from industrial mining effluents. The modified beads showed a 3-fold increase in ion adsorption and pose as excellent candidates for the manufacturing of three-dimensional (3-D) column filters for large-volume, high flux water treatment under atmospheric pressure.Biobased adsorbents and membranes offer advantages related to resource efficiency, safety, and fast kinetics but have challenges related to their reusability and water flux. Nanocellulose/alginate composite hydrogel beads were successfully prepared with a diameter of about 3–4 mm and porosity as high as 99%. The beads were further modified with  Biobased adsorbent beads with anionic charge used in three-dimensional (3-D) column filters for large-volume, high flux water treatment under atmospheric pressure. Literature reports show the successful use of inorganic materials 6–11 thanks to their meritorious properties, such as low environmental impact, high natural abundancy, and versatile surface chemistry which allows for functionalization.6,7 Cellulose offers an abundance of hydroxyl groups (–OH) which are susceptible to modification. An efficient method to introduce carboxyl groups (–COO–) and increase the affinity of cellulose towards cationic species is by selectively oxidizing the primary alcohol of its structure via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) mediated oxidation12 Currently, TEMPO oxidation constitutes the most used pretreatment method  for the preparation of highly charged CNF (often denoted as TEMPO-CNF or TO-CNF) and has been reported as a successful in situ modification method of cellulosic membranes.13 Despite the broad use of cellulose membranes in water purification, there are certain limitations in their capabilities.Cellulose and nanocellulose ) have emerged in recent years as versatile biobased choices for water treatment14–17 Such particles were successfully prepared from several different hybrid materials, including graphene/cellulose, poly(sodium acrylate)/cellulose, and starch mixtures, and proven efficient in water purification.18–20 The advantages of these adsorbents, often denoted as aerogel or hydrogel beads, are that they offer a higher number of functional groups per area compared to membranes, can be easily removed after water treatment, and can be packed in cylindrical configurations for the manufacturing of filters that resemble the ion exchange resins and allow high water permeance.The typically dense structure of the bodies of membranes leads to a high-pressure drop when used in dead-end flow and cross-flow. Pressure drop is defined as the pressure difference between the inlet and the outlet of a filter and it can result in the movement or even fracture of the filter. One way to reduce the pressure drop is to increase the permeability of a membrane either by reducing its thickness or increasing its porosity. However, both approaches involve a tradeoff between the mechanical properties and the efficiency of the filter. A less dense membrane matrix entails that a smaller amount of material is used and fewer functional groups are present to facilitate the adsorption. To overcome these drawbacks, 3-D adsorbents have been developed, for instance in the form of spherically shaped and porous aerogel and hydrogel particles.21–24 Alginate or alginic acid ) is a linear polysaccharide extracted from brown algae.21 It consists of blocks of -linked β-d-mannuronate (M) and α-l-guluronate (G) residues and as a copolymer, it is composed of either consecutive G or M units, for instance, GGGG or MMMM, or alternating M and G units, for instance, GMGMGM. A facile method to prepare highly porous fully biobased beads is with the use of alginate/cellulose hydrogel composites.2 solutions they instantly form spherical-shaped hydrogels because of the ionic-crosslinking of the G units of adjacent polymer chains, forming a so-called egg-box structure.6 Notably, it is believed that only the G units bind to Ca2+ because of their higher degree of coordination.21Due to its structure, alginate forms hydrogels upon interaction with ionic cross-linking agents such as divalent cations. When aqueous alginate solutions are poured dropwise into CaCl25,26 as well as cellulose/alginate beads27,28 have already been reported for heavy metal ion removal.The carboxyl and hydroxyl groups of alginate and cellulose make cellulose/alginate composites highly promising for use in water treatment applications for the removal of cationic pollutants. Stand-alone alginatein situ TEMPO oxidation on nanocellulose/alginate composite beads to control and enhance the performance which has not explored to date. We hypothesize that by oxidizing the available hydroxyl groups of cellulose, the amount of carboxyl groups would increase and thereby improve the removal capacity of the beads. For this purpose, commercial-grade defibrillated cellulose and SA were used for the preparation of beads which were then modified in situ by TEMPO-mediated oxidation. The effect of this modification was monitored by comparing the adsorption capacity of the pristine hydrogel beads (denoted as C-SA) with that of TEMPO-oxidized beads (denoted as TO-C-SA) toward Methylene Blue  and metal ion (Cd2+ from mining effluent), respectively.We aimed to investigate the effect of 2O) was used as received from Sigma-Aldrich. Sodium hypochlorite and hydrochloric acid were purchased from VWR. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) was purchased from TCI. All chemicals were used as received. Samples from Vormbäcken in Sweden, a water recipient of mining operation effluents, were kindly provided by Boliden.Commercial grade defibrillated cellulose with micro- and nanosized fibrils ) was supplied by Borregaard AB . Alginic acid sodium salt (SA), calcium chloride, sodium bromide, cadmium nitrate tetrahydrate, ethanol (EtOH 95%), and Methylene Blue (MB) were purchased from Sigma-Aldrich. Poly  cellulose dispersion in a 1 : 1 ratio for 1 h. The mixture was then collected with a syringe and was poured dropwise into a CaCl2 (0.7 M) solution, through a 2 mm needle  using a syringe pump at a rate of 0.5 mL min−1  was vigorously mixed in 100 mL distilled H2O. Then, 4.5 mL NaClO (aq)  was added and the pH of the reaction mixture was adjusted to 10 with dilute HCl (aq). Then, 10 g of partially dried hydrogel beads were added to the reaction mixture for 5 min. The beads were removed and transferred to an EtOH : H2O (1 : 4 ratio) mixture to quench the reaction for 1 min. Finally, the beads were washed with H2O and stored in H2O in the fridge until further use.TEMPO  was dispersed and 0.5 g NaBr (5 mM) was dissolved in 100 mL H−1, through 64 scans, and a resolution of 4 cm−1 with an ATR-FTIR spectrometer . All obtained spectra were baseline-corrected.Fourier transfer infrared spectroscopy (FTIR) spectra of lyophilized beads were recorded in the range of 400–4000 cm2O. The mixture was vigorously mixed with UltraTurrax (IKA) at 15 000 rpm until a homogeneous dispersion was obtained. Aliquots (1 mL) of the dispersions  were diluted with Milli-Q H2O to 100 mL and titrated with PDADMAC using a Stabino (ParticleMetrix) system. The charge density of PDADMAC is 0.307 μeq mL−1. The measurements were replicated three times for each sample and the surface charge densities were calculated according to V0 is the volume of titrant consumed to neutralize the sample, Vsample is the volume of the sample, and x is the concentration of the sample.Beads (1 g) were dispersed in 100 mL Milli-Q HImages of the surface of the prepared beads were recorded with a USB optical microscope . Moreover, the morphology of the surface and the cross-section of lyophilized beads were observed using a scanning electron microscope  with an acceleration voltage of 5 kV.23Ww and Wd are the weights in grams of the wet and dry beads, respectively, and ρm and ρw are the densities of the bead matrix and water, respectively, in g cm−3.The porosity of the beads was calculated by the gravimetric method using −1) were prepared. Beads (1 g) were immersed in 100 mL dye solutions and aliquots were removed at specific times . The aliquots were then analyzed with UV-vis spectroscopy  in the spectral range 200–800 nm and the maximum absorbance at 665 nm (corresponding to MB) was recorded.Aqueous solutions of MB (5 mg L2+ (400 mg L−1) were prepared. Beads (1 g) were immersed in 100 mL Cd2+ solutions and left for 1 h. The beads were then collected and dried at 40 °C under vacuum. Elemental analysis of the beads was performed with energy-dispersive X-ray spectroscopy (EDS) using a scanning electron microscope equipped with an X-ray detector , at an acceleration voltage of 15 kV.Aqueous solutions of Cd2+ solution was performed via Thermo Fisher (K-Alpha) with micro-focused Al Kα radiation (energy of 1486.6 eV). A carbon peak at a binding energy of 284.2 eV was used as a reference. For these experiments, samples from effluents of a mining industry were used to study the selectivity of the beads towards Cd2+.Similarly, X-ray photoelectron spectroscopy (XPS) analysis of beads after interaction with Cd2O to pass through a chromatographic column with a beaded rim  was recorded. The measurement was repeated with the use of commercial filter paper  and 1, 5, and 10 g of the beads. The reported values are averages of 5 replicates.The time needed for 100 mL Hin situ modification were characterized with FT-IR (−1 which correspond to stretching vibrations of – –COO− (asymmetric), –COO− (symmetric), and C–O bonds, respectively.24 The increase in the intensity of the peaks of TO-C-SA compared to C-SA is attributed to the increase of carboxyl groups after oxidation.Functional groups of the prepared beads before and after the th FT-IR . The rec−1 for the C-SA to 477 ± 47 mmol kg−1 for TO-C-SA. An earlier report from our group on in situ TEMPO oxidation of cellulose membranes , showed an increase in acidic group content from ∼7 to ∼42 mmol kg−1, corresponding to a significant 6-fold charge increase.13 In the current case, the charge density of the beads after modification is significantly higher , however, the increase between modified and unmodified beads showed only a two-fold increase which can be attributed to the abundance of carboxyl groups in the alginate phase used for beads processing. The concept of bead formation by nanocellulose and alginate by Ca2+ crosslinking and subsequent TEMPO oxidation is schematically shown in The increase in the number of carboxyl groups in the composition of the beads after oxidation is also supported by an increase in surface charge density from 291 ± 51 mmol kgThe morphology of the beads was monitored with a digital optical microscope, as well as SEM imaging for their surface and cross-section .in situ oxidation. The average dry weight of the TO-C-SA beads was about half of the C-SA beads while their average wet weight was almost identical. This is probably attributable to the fact that the modified beads are more hydrophilic and, therefore, absorb more H2O.12 TO-C-SA contains more carboxyl groups than C-SA, explaining the difference in hydrophilicity. Nevertheless, the differences in average dry and wet weights did not significantly affect the porosity of the beads before and after the modification . In situ oxidation of the beads increased the total MB removal efficiency, reaching 91% after 12 h  was also reported for MG adsorption.31 These composite beads exhibit good adsorption efficiencies. However, it contains inorganic clays e.g. montmorillonite or halloysite that can be a source of contamination for long-term exposure. Our beads are pure organic biopolymers without any inorganic materials with good efficiency. It should be also notted that most of these efficiencies are based on the biopolymer of the beads e.g., chitosan.31 Cellulose-based beads are economically cheaper than chitosan based beads.32 Copper ions were suggested to replace Ca2+ for the formation of Graphene oxide (GO)/SA/Carrageenan (GO/Alg-Car) beads (Ala : Car : GO = 2 : 2 : 1).33 The use of Ca2+ is environmentally benign compared to Cu2+ ions that can be released into the aqueoutic system causing secondary pollution. SA beads of meso-tetrakisporphyrinto) zinc(ii) complex (Zn(TMP)) containing 3% of SA was reported for MB adsorption.34 TO-C-SA beads exhibit comparable adsorption efficiency with a suitable equilibrium time to reach the steady state  from 2.53 to 6.98  of 284.4, 286.6, 287.2, and 288.2 eV which correspond to C–C, C–O, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, and O–CO, respectively  for alginate (G or M) and TEMPO-oxidized cellulose (O–CO), respectively.37The deconvolution of the high-resolution C 1s spectrum of C-SA beads after Cdectively . Similarectively . The fir2+ adsorption reveals peaks at BE of 530.9, 531.9, 532.9, and 533.9 eV, which correspond to C–O, CO, O–CO, and O–Cd, respectively . The dewatering time increased to 46.3 ± 2.6 s with the use of commercial-grade filter paper. The required dewatering time was 28.8 ± 2.2, 30.5 ± 3.1, and 44.2 ± 3.6 s when packing the column with 1, 5, and 10 g of C-SA beads, respectively  The required dewatering time was identical when instead filling the column with TO-C-SA beads was 28.6 ± 1.1, 30.4 ± 0.7, and 44.0 ± 2.5 s for 1, 5, and 10 g of beads, respectively , EDS, and XPS (for Cd2+). Interestingly, the EDS elemental analysis of the beads showed an almost 3-fold improvement in Cd2+ removal after the in situ oxidation. Furthermore, the obtained water flux data indicates that the prepared beads can be used for the manufacturing of column filters. This approach offers filters with a higher amount of adsorbent and better water permeability than 2D membrane filters. Overall, the abundance of the component feedstock, as well as the ease and efficiency of the modification route indicate that the nanocomposite beads are promising candidates for larger production and manufacturing of column filters for water purification. Nevertheless, how the mechanical properties of the beads were affected by the oxidation and how the presence of several charged contaminants in the same sample affects the adsorption performance of the beads will be a subject of future investigation. The long-term stability and recyclability of the beads will also be a topic of further investigation.We devised a facile method of preparing nanocellulose/alginate composite beads with increased adsorption performance derived from the D. G. methodology, data collection, investigation, writing-original draft, D. G.; investigation, writing-review and editing; H. N. A., methodology, data collection, investigation, writing-original draft, D. G.; investigation, writing-review, and editing; A. P. M., conceptualization, supervision, writing-review and editing; U. E., supervision, writing-review and editing. All authors have read and agreed to the published version of the manuscript.There are no conflicts to declare.NA-005-D3NA00531C-s001