Furthermore, for lime with a particle size of 20 mm and a calcination rate of 0%, the thickest layer of 2CaO·SiO2 was observed after 120 s of dissolution in slag A2 at 1400 °C. Overall, the dissolution rates of lime with different particle sizes in slag A1 to A4 showed a gradual increase. On the other hand, the dissolution rates of lime with different calcination rates in slag A1 to A4 exhibited an initial increase, followed by a decrease, and then another increase. The formation of a high-melting point and continuous dense 2CaO·SiO2 layer during the dissolution process hindered the mass transfer between lime and slag.China’s 2022 crude steel production soared to an impressive 1.018 billion tons, and steel slag constituted approximately 10% to 15% of this massive output. However, a notable hindrance to the comprehensive utilization of steel slag arises from the fact that it contains 10% to 20% of free calcium oxide (f-CaO), resulting in volume instability. To address this challenge, our study delved into the dynamic transformation of the interface between lime and slag, as well as the fluctuations in the dissolution rate of lime. An Electron Probe Micro Analyzer, equipped with an energy-dispersive spectrometer, was employed for the analysis. Our findings revealed that the configuration of the reaction interface between quicklime and slag underwent alterations throughout various phases of converter smelting. At a temperature of 1400 °C, several significant transformations occurred, including the formation of a CaO-FeO solid solution,  olivine, and low-melting point  silicate minerals. With the gradual reduction in FeO content, a robust and high-melting 2CaO·SiO In 2022, China achieved a momentous feat in its crude steel production, reaching an astounding 1.018 billion tons. However, given its status as a substantial consumer and emitter of fossil fuels, the iron and steel industry has become a focal point for endeavors aimed at reducing carbon emissions .2O3-based slag to investigate the distribution of slag components proximate to the interface and the formation of 2CaO·SiO2, referred to as C2S hereafter. Interestingly, the precipitation of C2S was observed at a distance from the interface. In parallel research, D. Sichen et al. [2 and CaO-FeO-SiO2 slags. Their work emphasized that lime exhibited a heightened solubility within the FeO-SiO2 system, while in the CaO-FeO-SiO2 system, a dense layer of C2S emerged. This phenomenon was attributed to the increased dissolution driving force of CaO within the FeO-SiO2 system, and the formation of the C2S layer was exclusively observed under conditions of low FeO content. Furthermore, Xia et al. [2O5. Their investigation revealed that in the CaO-FeO-SiO2-P2O5 system, the interface exhibited the formation of a solid solution composed of 3CaO·P2O5 instead of the customary C2S layer. However, it is noteworthy that the creation of this solid solution also impeded the dissolution of lime. Lastly, Ludimila et al. [The advancement of expeditious lime dissolution and its integration into slag represents a highly significant and extensively researched domain. Matsushima et al.  conducten et al.  delved ia et al.  exploreda et al.  embarkedThe development of a compact C2S layer hinders the dissolution of lime by deteriorating the kinetic conditions, ultimately leading to a surge in undissolved lime particles during the latter phases of converter slag processing. This limitation presents a challenge for achieving high recycling rates of undissolved lime particles in converter slag. The dissolution of lime is influenced by both the physical structure of lime itself and the properties of the slag ,4,5,6,7.3, with a content of 98.55%. It also contains small amounts of MgO, SiO2, and other components. X-ray diffraction analysis using a PHILIPS-Xpertpro instrument confirmed that calcite (CaCO3) is the only mineral phase present in the limestone. The slag system studied in this experiment corresponds to the early to middle stage of the slag-forming route based on the CaO component in converter smelting, with the slag composition ratios shown in 2, and MgO were used as chemically pure reagents, while FeO was added in the form of ferrous oxalate. The corresponding points of the synthetic slag composition in the ternary phase diagram are shown in The raw material used in this experiment is laboratory-calcined quicklime, which is derived from limestone provided by a large domestic steel company. The composition of the limestone was determined by using X-ray fluorescence (XRF) analysis, which showed that the main component of the limestone is CaCOThe experimental procedure encompasses several sequential stages, including the preparation of cylindrical lime, synthesis of the slag, dissolution experiments, and sample analysis. The initial step involves the preparation of cylindrical limestone particles, and the detailed process is as follows: First, raw limestone was initially sliced into small pieces using a cutting machine. Subsequently, the limestone blocks were ground into cylindrical shapes, each with heights of 15 mm and diameters ranging from 10 mm, 15 mm, 20 mm, to 25 mm, respectively, utilizing a grinding machine. These cylindrical limestone specimens were then meticulously cleansed and subjected to drying in an oven at a temperature of 200 °C for a duration of three hours. After the drying process, the limestone samples were allowed to cool to room temperature and were preserved in a vacuum container for future use. The next step involved the utilization of a carbon tube furnace, with high-purity N2 gas serving as a protective atmosphere. This furnace was gradually heated at a specified heating rate until it reached a temperature of 1350 °C. The previously prepared cylindrical lime particles were placed within a molybdenum basket, strategically located within the constant temperature zone of the carbon tube furnace. Initially, all lime particles, regardless of their size, were subjected to a 10 min heating period to facilitate the initial formation. Subsequently, the 15 mm lime particles underwent distinct heating durations to achieve varying calcination degrees, specifically 0%, 44%, 60%, and 84%, respectively. Finally, the calcined lime was cooled to room temperature and stored in a vacuum desiccator for subsequent use. The calcination rate is defined according to Equation (1):2 gas, flowing at a rate of 10 L/min, was introduced into the furnace to maintain a protective atmosphere. The temperature within the furnace was then gradually increased at a rate of 10 °C/min until it reached the target temperature of 1400 °C. Upon reaching this temperature, the system was allowed to equilibrate for 5 min, ensuring the complete melting of the slag. Subsequently, the prepared lime was introduced into the molten slag. After a dissolution period of 120 s, the lime particles were carefully extracted from the slag and rapidly cooled to room temperature through air cooling for further analysis.In the second phase of the experiment, the synthetic slag was meticulously prepared in accordance with the composition outlined in The concluding step of the experiment entails the analysis of the lime samples. The cooled lime samples were cut in half, partially embedded using a mounting press, and then subjected to grinding, polishing, and cleaning with an ultrasonic cleaning device. Subsequently, the samples were dried in a vacuum desiccator and underwent gold sputtering for scanning electron microscopy observation of the microstructure at the lime–slag reaction interface. The collected samples were analyzed using a field emission electron probe microanalyzer (EPMA 8050G) to observe the microstructure and a configured energy dispersive spectrometer (AZtecLive UltimMax 40) to determine the composition of the microregions. The average thickness of each phase layer in the slag was measured using ImageJ software.The variation in lime particle diameter after dissolution in slag for different particle sizes and calcination rates is presented in The dissolution rate of quicklime in the slag, denoted as interval . This ra−6 m/s. In slags A1, A3, and A4, the dissolution rate of lime particles showed a trend of rapid increase, followed by a slow increase, and then another rapid increase with the increase in particle size. Specifically, in slag A1, the dissolution rate of lime increased gradually from 1.44 × 10−6 m/s to 3.33 × 10−6 m/s as the particle size increased. In slag A3, the dissolution rate increased from 2.51 × 10−6 m/s to 4.37 × 10−6 m/s, and in slag A4, it increased from 1.39 × 10−6 m/s to 3.51 × 10−6 m/s. When the lime particle size was 10 mm, the maximum dissolution rate was observed in slag A2, with a value of 2.51 × 10−6 m/s. When the lime particle size was 15 mm, the dissolution rates in different slags were relatively close, with the maximum rate of 2.51 × 10−6 m/s in slag A3 and the minimum rate of 1.44 × 10−6 m/s in slag A1. The largest difference in dissolution rates occurred when the lime particle size was 20 mm. In this case, the dissolution rates in the slags followed the order of A2 > A3 > A4 > A1, and the difference between the maximum and minimum rates was 0.8 × 10−6 m/s. When the lime particle size was 25 mm, the dissolution rates in the slags were in the order of A3 > A2 > A4 > A1, with a maximum rate of 4.37 × 10−6 m/s. From −6 m/s, 2.59 × 10−6 m/s, and 4.30 × 10−6 m/s, respectively. However, in slag A2, the lime dissolution rate reached its maximum value of 2.91 × 10−6 m/s at a calcination rate of 44%. Additionally, the growth in lime dissolution rate was most significant in the range of 60% to 84% calcination rate. Among them, when lime with an 84% calcination rate was dissolved in slag A3, it exhibited the highest growth in dissolution rate, increasing from 1.00 × 10−6 m/s to 4.30 × 10−6 m/s. The order of lime dissolution rates in this case was A3 > A2 > A4 > A1, which followed the same pattern observed for lime particles with a particle size of 25 mm.By substituting the data from The evolution of the average thickness of the  olivine layer formed at the lime–slag reaction interface during the 1400 °C reaction, involving lime particles of different sizes and various slags, is graphically represented in The microstructure of the reaction interface when lime particles with a diameter of 10 mm reacted with different slags at 1400 °C is shown in As lime with varying calcination rates interacted with different slags at 1400 °C, the average thickness of the  olivine layer formed at the lime–slag reaction interface exhibited fluctuations with the rise in calcination rate, as depicted in 2-MgO-based slag system at 1350 °C. Upon introducing rapidly calcined quicklime into the slag, the liquid slag envelops the lime particles. It infiltrates the interior of the lime through cracks and micropores formed during calcination, initiating the lime–slag interface reaction [2 is impeded by this solid solution layer. Within the solid solution layer, CaO diffuses into the slag and engages in reactions with MgO, FeO, and SiO2 present in the slag. This results in the formation of  olivine and  olivine. Since magnesium olivine and iron olivine can create infinite solid solutions, the chemical formula of olivine can be simplified as 3O∙SiO2. As the lime–slag interface reaction progresses, concentration gradients materialize. The concentration of FeO increases from the slag to the  olivine phase, then to the CaO-FeO solid solution, ultimately becoming concentrated in the CaO-FeO solid solution layer. This concentration disparity acts as a driving force, prompting the diffusion of FeO from the slag and the CaO-FeO solid solution layer into the  olivine phase and lime, respectively. Furthermore, the CaO concentration gradient, arising from the reaction between lime and slag, facilitates the diffusion of CaO from lime to the slag, thereby perpetuating the reaction. When the slag has a high SiO2 content, all the free SiO2 in the slag cannot react completely with the CaO provided by lime. In this case, CaO reacts with the formed  olivine, resulting in the formation of low-melting-point calcium–magnesium silicate phases containing MgO in the slag [2, the quantity of  olivine within the slag gradually increases, leading to a progressive thickening of the solid solution layer. Simultaneously, lime continues to dissolve in the slag, which causes an uptick in the CaO content within the slag. This, in turn, prompts reactions with FeO, resulting in the formation of additional solid solutions and consequently leading to a decrease in the FeO content. It is important to note that CaO not only reacts with SiO2 to generate various silicates but also, when the FeO content is below 20%, continues to replace MgO in the calcium–magnesium silicate phase, forming a dense C2S with a melting point as high as 2130 °C [The schematic representation in reaction ,10,11. Dthe slag ,13,14. A 2130 °C ,16. The  2130 °C ,18,19,20k) can be determined using the Formula (5) developed by Matsushima et al. [k is the liquid-phase mass transfer coefficient in mm/s, 3), 3), and ∆(mol%CaO) is the driving force for dissolution. It quantifies the disparity between the initial CaO concentration and the saturation CaO concentration. The ∆(mol%CaO) values for various slags are provided in CaO value can be ascertained by analyzing the liquidus line within the CaO-FeO-SiO2-MgO phase diagram [The liquid-phase mass transfer coefficient . The results are shown in −6 m/s during 120 s of dissolution in the slag. Similarly, the lime with an 84% calcination rate also exhibits the highest average mass transfer coefficient of 20.15 × 10−6 m/s during the same dissolution time in the slag.Based on the data in (1)In the course of the reaction between quicklime and slag, several key transformations take place. These include the development of a CaO-FeO solid solution, the formation of  olivine, and the generation of low-melting-point calcium–magnesium silicate minerals containing MgO. As the FeO content in the system diminishes, CaO engages in reactions with the  olivine, leading to the creation of a high-melting-point and compact C2S layer. This C2S layer effectively acts as a barrier, preventing the further dissolution of lime.(2)The dissolution rate of lime with different particle sizes in slags A1 to A4 generally shows an increasing trend at 1400 °C, reaching the maximum dissolution rate in slag A4. However, the dissolution rate of lime with different calcination rates in slags A1 to A4 initially increases, then decreases, and finally increases again. Therefore, it is necessary to select lime with an appropriate particle size and combustion rate to complete a rapid dissolution before A4 to avoid residual CaO entering the slag.(3)At 1400 °C, lime with a particle size of 20 mm exhibits the maximum average thickness of the C2S layer during 120 s of dissolution in slag A2, which is 12.11 μm. Lime with a calcination rate of 0% shows the maximum thickness of the generated C2S layer, which is 11.41 μm, during 120 s of dissolution in slag A2.(4)−6 m/s and 20.15 × 10−6 m/s, respectively.The average mass transfer coefficient for lime with different particle sizes and calcination rates during dissolution in slags A1 to A4 at 1400 °C gradually increases. The maximum values are achieved with a particle size of 25 mm and a calcination rate of 84%, measuring 30.41 × 10


The successful development of effective viral vaccines depends on well-known correlates of protection, high immunogenicity, acceptable safety criteria, low reactogenicity, and well-designed immune monitoring and serology. Virus-neutralizing antibodies are often a good correlate of protective immunity, and their serum concentration is a key parameter during the pre-clinical and clinical testing of vaccine candidates. Viruses are inherently infectious and potentially harmful, but we and others developed replication-defective SARS-CoV-2 virus-like-particles (VLPs) as surrogates for infection to quantitate neutralizing antibodies with appropriate target cells using a split enzyme-based approach. Here, we show that SARS-CoV-2 and Epstein–Barr virus (EBV)-derived VLPs associate and fuse with extracellular vesicles in a highly specific manner, mediated by the respective viral fusion proteins and their corresponding host receptors. We highlight the capacity of virus-neutralizing antibodies to interfere with this interaction and demonstrate a potent application using this technology. To overcome the common limitations of most virus neutralization tests, we developed a quick in vitro diagnostic assay based on the fusion of SARS-CoV-2 VLPs with susceptible vesicles to quantitate neutralizing antibodies without the need for infectious viruses or living cells. We validated this method by testing a set of COVID-19 patient serum samples, correlated the results with those of a conventional test, and found good sensitivity and specificity. Furthermore, we demonstrate that this serological assay can be adapted to a human herpesvirus, EBV, and possibly other enveloped viruses. Vaccines are among the most successful and efficient means to counteract infectious diseases. Prophylactic immunizations effectively prevent millions of deaths and serious diseases worldwide every year and substantially contributed to the eradication of harmful viruses in the past . VaccineConventional VNTs (cVNTs) rely on replication-competent, infectious viruses, permissive target cells and often on in vitro cytopathic effects (CPEs) that appear as foci or plaques several days after infection, reflecting the number of infectious viruses. Depending on the individual virus, CPE counting can be cumbersome or even impossible without additional methods, such as immunostaining, limiting dilution approaches, or PCR, to quantify viral infection . All cVNCoronaviridae. SARS-CoV-2 virions contain a positive-sense, single-stranded RNA genome of almost 30 kb, which codes for four structural proteins, spike (S), envelope (E), membrane (M), and nucleoprotein (N), and several accessory proteins, which have important roles in viral replication or immune modulation ..+ VLP anus virus D,E 29]..+ VLP anus virus , and it We also demonstrate VLP–EV co-association and fusion with another enveloped virus, EBV, which differs from SARS-CoV-2 in many aspects. We successfully used EB-VLPs to quantitate virus-neutralizing antibodies in a cell-free and cell-based VLPNT. NmAbs directed against EBV gp42 or gp350 and serum samples from EBV-seropositive donors specifically neutralized EB-VLP in both assays, confirming the potential of this technology.In summary, we show that VLPs co-associate and fuse with permissive EVs in vitro and demonstrate that this process can be exploited as a new principle for diagnostic VNTs. Speculating whether the fusion of viruses with EVs also occurs in infected individuals in vivo remains tempting.


The excessive presence of single-use plastics is rapidly degrading our natural environment on a global scale due to their inherent resistance to decomposition. Wet wipes used for personal or household purposes contribute significantly to the accumulation of plastic waste. One potential solution to address this problem involves developing eco-friendly materials that possess the ability to degrade naturally while retaining their washing capabilities. For this purpose, the beads from sodium alginate, gellan gum, and a mixture of these natural polymers containing surfactant were produced using the ionotropic gelation method. Stability studies of the beads by observing their appearance and diameter were performed after incubation in solutions of different pH values. The images showed that macroparticles were reduced in size in an acidic medium and swelled in solution of pH-neutral phosphate-buffered saline. Moreover, all the beads first swelled and then degraded in alkaline conditions. The beads based on gellan gum and combining both polymers were the least sensitive to pH changes. The compression tests revealed that the stiffness of all macroparticles decreased with the increasing pH of the solutions in which they were immersed. The studied beads were more rigid in an acidic solution than in alkaline conditions. The biodegradation of macroparticles was assessed using a respirometric method in soil and seawater. It is important to note that the macroparticles degraded more rapidly in soil than in seawater. Single-use plastics (SUPs) were invented for modern society and are commonly used fast-moving consumer products thrown away after a single use . PlasticSeveral natural polymers have been widely used as polymer matrices to develop functional beads. The polymer beads generally consist of the polymer matrix and loaded active substance. Sodium alginate (ALG) is an anionic polysaccharide obtained from brown algae, which is composed of two structural units of 1–4 linked α-L-guluronic acid and β-D-mannuronic acid . One of Another natural polymer used to prepare beads is gellan gum. Gellan gum (GG) is a bacterial exopolysaccharide that contains repeating units of β-D-glucose, L-rhamnose, and D-glucuronic acid . Gellan There are several studies about the combination of alginates and gellan gum. Park et al. produced Pseudomonas-aeruginosa-encapsulated alginate/gellan gum microbeads to investigate the biodegradation of diesel-contaminated groundwater. The results confirmed that encapsulation could protect microorganisms from toxic contaminants, and the activity of microorganisms could be maintained with the protective barrier of biodegradable molecules . The comThe creation of cleaning wipes involves the addition of surfactants to the materials. Alkyl polyglucosides are non-ionic surfactants consisting of a hydrophilic sugar moiety linked to a hydrophobic alkyl chain. They are produced from renewable raw materials, such as fatty alcohols  and glucose  ,26. DecyOur research aimed to produce biodegradable polymer beads containing surfactant (decyl glucoside), which could be a component of eco-friendly wet wipes. Sodium alginate, gellan gum, and a mixture of both natural polymers were used to prepare macroparticles with an inotropic gelation method and calcium chloride as a crosslinker. Although both biopolymers were used to produce the beads earlier, in this experiment, we used them to entrap the surfactant, which has not been tested before. The obtained beads in their wet form and after immersion in different pH solutions were characterized by compression tests to determine Young’s modulus according to Hertz’s approach. The influence of pH on the structural integrity of the beads were defined by exploring their stiffness after exposure to solutions with diverse pHs. The stability of the samples in solutions of different pHs was examined owing to various applications of the beads with a surfactant in products that come into contact with the skin or in household chemicals. This analysis aimed to assess the effects of pH variations on maintaining the bead integrity and functionality, which are crucial for ensuring their efficacy. The investigation involved subjecting the beads to a pH ranging from acidic to alkaline solutions, simulating scenarios where the beads might encounter different environmental conditions. By monitoring changes in the bead appearance and diameter by optical microscope under varying pH conditions, valuable insights were gained into their behavior and potential applications. Biodegradation in soil and seawater of the macroparticles was determined using a respirometric method by OxiTop apparatus, which measures the rate of oxygen consumption by microorganisms  to indicate their metabolic activity during the degradation process.3/g and a = 1 [2), acetic acid (CH3COOH), sodium acetate (CH3COONa), disodium phosphate (Na2HPO4), monosodium phosphate (NaH2PO4), sodium carbonate (Na2CO3), and sodium bicarbonate (NaHCO3) were supplied by Chempur . Decyl glucoside (DG) was acquired from Greenaction . All used chemicals were of analytical grade.Sodium alginate (ALG) was supplied by BÜCHI Labortechnik AG , for which the viscosity average molecular weight was determined in our laboratory, and it was equal to 55,800 for K = 0.0178 cmnd a = 1 . Gellan w/w), sodium alginate solution (1.5% w/w), and a mixture of gellan gum (0.75% w/w) with sodium alginate (0.75% w/w) were prepared. The surfactant (1% w/w) was added to all solutions. Deionized water was the solvent of all components. Next, beads were formed by dripping the polymer solutions from a syringe (diameter of 1.2 mm) to 0.5 M CaCl2 solution under constant stirring. The macroparticles were kept in a crosslinking solution for 1 h. Then, they were washed 4 times with deionized water. Three types of beads containing washing agents were produced: beads made of sodium alginate (ALG), gellan gum (GG), and sodium alginate/gellan gum (ALG + GG) mixture.The microspheres were produced from sodium alginate and gellan gum with incorporated surfactant (Lauryl Glucoside) by the inotropic gelation method. First, gellan gum solution (1.5% w/v) NaHCO3 solution (pH = 9), and 1% (w/v) Na2CO3 solution (pH = 10) to study the stability of the prepared macroparticles. The obtained polymer beads were immersed in these solutions for 2, 4, and 24 h. After each time, the appearance of macroparticles was observed by the optical microscope Motic SMZ-171 BLED  at a magnification of ×10. The diameter of the beads was measured using this microscope. The images and sizes of the beads stored in the deionized water were also obtained.The solutions of different pH were prepared: acetate buffer , phosphate buffer , 1% . The beads soaked in solutions of different pH (pH = 4–10) for 2 h were examined. The tests were carried out at a 1 mm/min compression speed. The Hertz theory was used to determine Young’s modulus. Hertz’s model describes the relationship between force and displacement for an elastic sphere compressed between two flat smooth surfaces, according to the following equation:lacement . E* is Ho be 0.5 .The results were recorded using the Trapezium X software . The presented data are the average values calculated from 7 measurements for each type of bead.The prepared bead biodegradation in soil and seawater was determined using a respirometric method with a OxiTop Control OC 110 set , which analyzed the microbial respiration activity (oxygen uptake).2 absorber (0.4 g NaOH) were added to the bottles. The samples were incubated at 20 °C for 28 days. Seawater was used as a control sample (endogenous respiration). The respiratory activity of microorganisms was demonstrated in mg O2 × dm−3 of seawater after 28 days.The biological oxygen demand (BOD) measurement with OxiTop-Control in seawater was performed according to the supplier’s operating instructions. The seawater was collected from the Baltic Sea near Gdansk. The seawater (164 mL) and each type of polymer bead (10 g) were put in 500 mL glass bottles. Nitrification solution inhibitor NTH 600 (3 drops) and the carrier containing CO2 absorber (0.4 g NaOH) were put in 1 dm3 glass containers. The specimens were incubated at 20 °C for 28 days. The neat soil was used as a control sample (endogenous respiration). Respiratory microbial activity was expressed as mg O2 × kg−1 of soil after 28 days. The oxygen consumption measurements in seawater and soil for all samples were recorded every two days.In the case of biodegradation of the beads in soil, the measured values were also recorded by the OxiTop-Control system in the pressure p mode. The soil (100 g), polymer macroparticles (10 g) and a carrier with CO+ interacts with only one carboxyl group of the alginate chain, the Ca2+ interacts ionically with the carboxyl group of guluronate residues, forming a three-dimensional network usually described by the egg box model [The polymer beads based on gellan gum and sodium alginate were formed by ionic crosslinking. The gelation of gellan gum occurs through an ionic chemical bonding between calcium cations and two carboxylate groups from glucuronic acid units in the gellan gum chains. Hydrogen ions from the gellan gum are exchanged with calcium ions ,33. The ox model .The obtained polymer beads loaded with the surface active agent were immersed in solutions of different pH for 2, 4, and 24 h. The appearance and diameter of these macroparticles were observed and measured using the optical microscope, which was shown in The stability of beads in solutions at different pHs is crucial for their successful application in skincare or personal hygiene products. When these beads come into contact with the skin, it is important that they remain intact and do not degrade too quickly or release their active ingredients when mechanically damaged (after being pressed). These beads can effectively provide their intended benefits and enhance the overall user experience by ensuring stability across a range of pH, from acidic to alkaline.Similarly, materials used in cleaning agents for domestic use should indicate the appropriate pH values for optimal performance in removing dirt and other contaminants. The pH of a cleaning product can significantly impact its cleaning efficiency. By indicating the appropriate pH range on the packaging, consumers can make informed decisions about the products they choose for different cleaning tasks.Products containing acidic agents are particularly effective in dissolving limescale and rust. These agents break down the mineral deposits making them easier to remove. Acidic cleaning products are commonly used in bathrooms, kitchens, and other areas prone to the buildup of these substances.On the other hand, alkaline products are specifically formulated to dissolve fats and grease. Alkaline agents have the ability to break down the chemical bonds in fatty substances, allowing them to be easily washed away. This makes alkaline cleaning products well-suited for tackling greasy kitchen surfaces, oven grime, and other areas where stubborn grease accumulations are common.In summary, understanding the appropriate pH range for different applications is crucial for developing and using materials, whether for skincare products, cleaning agents, or other purposes. Considering pH compatibility and selecting the right products can ensure optimal performance, effectiveness, and user satisfaction.2+ migration into the solution or by strengthening the gel network by diffusing H+ ions [The presented images indicate that in the case of beads’ immersion in acidic solution, the macroparticles were reduced in size compared to the initial samples stored in deionized water , Table 1 H+ ions . MaintaiIt was also noticed that the prepared spheres swelled in phosphate-buffered saline of pH-neutral. The alginate beads showed the highest efficiency of swelling, and this process was more effective in solutions of higher pH . The sweIn alkaline solutions, all the samples first swelled and then degraded , Table 1It should be emphasized that swelling supports degradation processes in the case of the prepared polymer beads. Swelling in polymers refers to the absorption of a solvent or liquid, causing an increase in the polymer’s volume . SwellinThe mechanical testing results of the obtained polymer beads containing surfactant are presented in − and Na+ ions. The beads prepared from the alginate and gellan gum mixture were characterized by the lowest values of elastic modulus after conditioning in pH 4 and 5 solutions, showing the destabilizing effect of mixing both polymers, whereas these beads had higher values of Young’s modulus than alginate beads in the remaining solutions, revealing stabilizing effect of mixing. It is worth noting that the alginate beads disintegrated after 2 h of immersion in the solution of pH = 9.As one can see in Chan et al. studied the mechanical behavior of alginate beads and demonstrated Young’s modulus values in the range between 250 and 800 kPa, depending on the bead formulation. Their measurement was performed at a high-speed compression (40 mm/min), and the concentrations of sodium alginate solution used to prepare beads varied from 5 to 50 g/L. It was found that gel beads were considered viscoelastic and could lose liquid under compression . In our 2 is readily available, aerobic heterotrophic microorganisms are primarily responsible for the degradation of complex materials, with microbial biomass, CO2, and H2O as the final products [2 by NaOH pellets [During the aerobic biodegradation process, when Oproducts ,45. Biol pellets . The bio pellets . Oxygen 2 × dm−3), while oxygen consumption observed for ALG and ALG + GG beads were much lower (about 180 mg O2 × dm−3). It was found that water microorganisms were characterized by higher metabolic activity (higher oxygen demand) in the presence of ALG macroparticles compared to GG macroparticles.The data presented in 2 × kg−1), while oxygen uptake recorded during the decomposition of ALG + GG and GG beads was similar (approx. 520 mg O2 × kg−1). It should be emphasized that the obtained polymer macroparticles degraded more efficiently in soil than in seawater. Summing up, the beads based on sodium alginate and gellan gum containing washing agent biodegraded during the 28 days.The biodegradation in the soil of the studies macroparticles occurred differently. The highest oxygen consumption in soil was observed during the biodegradation of ALG beads (590 mg OThe biodegradation process of polymers in soil and seawater can differ due to variations in environmental factors and the presence of different biological and chemical agents. Both soil and seawater contain diverse microbial populations that can contribute to polymer degradation. However, the types of microorganisms present and their activity may differ. Soil tends to have a more diverse range of microorganisms, including bacteria, fungi, and archaea, which can enzymatically break down polymers. Seawater typically has a lower microbial diversity, primarily comprising bacteria and marine fungi, which may have specific enzymatic capabilities for polymer degradation in a marine environment ,49. In aThe biopolymer beads made of sodium alginate and gellan gum with the addition of a surfactant were obtained using the inotropic gelation technique. Decyl glucoside was used as the washing agent in this experiment. Based on the findings, it is evident that the beads prepared from gellan gum and the mixture of gellan gum and sodium alginate exhibit stability in solutions with pH values ranging from acidic to alkaline. This stability was observed even after 24 h of incubation, as the beads maintained their shape, integrity, and functionality.Moreover, the stiffness of all macroparticles decreased with the increasing pH of the immersed solutions due to the swelling. The tested samples were more rigid in an acidic than an alkaline medium. All prepared beads loaded with surfactant biodegraded after 28 days of incubation in soil and sea water. This biodegradability is a significant advantage, as it aligns with the global trend of seeking ecological solutions to combat the escalating plastic pollution crisis. The prepared biodegradable beads have the potential to serve as environmentally friendly components in wet wipes, offering both cleaning properties and eco-consciousness. These macroparticles will be suitable for application in products with acidic pH. Moreover, the beads based on gellan gum and a combination of gellan gum and sodium alginate can be utilized in alkaline products thanks to their stability even at pH = 10.The urgent need to reduce plastic consumption arises from the growing concern over plastic pollution and its accumulation in water environments and landfills. In response to these global challenges, developing ecological solutions such as these biodegradable beads present a promising approach. Incorporating such eco-friendly alternatives into various products can contribute to mitigating the detrimental impacts of plastic waste on the environment.


Bos taurus bulls (68.1%), older than seven years (50.0%), and those belonging to dairy farms (75.5%) had higher seropositivity compared to Bos indicus (17.7%), younger than seven years (42.2%), and those from beef farms (15.5%), respectively. Moreover, Bos taurus bulls had a higher risk of being seropositive than Bos indicus . BLV DNA was found in one semen sample  from a seropositive bull. The importance of serum and molecular BLV screening in semen samples and the potential role of some risk factors associated with the disease, such as the bull’s age, genotype, and type of livestock productive system, is argued in the present report.Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a persistent infection and the most important neoplastic disease in cattle. It is spread primarily by transferring infected lymphocytes through blood from carriers to healthy animals. The present study is aimed at determining the seropositivity of BLV in breeding bulls from Costa Rica and at detecting for the first time in the country BLV DNA in bull semen. Between May 2011 and August 2018, 379 blood and 133 semen samples were collected from bulls distributed in 118 farms. The serum was analyzed by an enzymatic immunoassay and the semen by polymerase chain reaction and sequencing. BLV seropositivity was 43.5% (165/379), while 64.4% (76/118) of the farms had positive reactors. Holstein (75.7%) and Jersey (73.0%) breeds showed the highest seropositivity. In addition,  Retroviridae family (genus Deltaretrovirus). This viral infection causes significant economic losses in the cattle industry worldwide  has also been reported (Hopkins and DiGiacomo Enzootic bovine leukosis (EBL) is the most important neoplastic disease in cattle caused by the bovine leukemia virus (BLV), which belongs to the Semen from infected bulls could represent a source of infection only if it contains lymphocytes, a common condition in seminal vesiculitis, and other infectious conditions from the urinary and reproductive tract  BLV seroprevalence was determined in breeding bulls from the South Pacific area , beef (n = 47), and dual-purpose (n = 30) production, distributed in the Dry Pacific and North Huetar regions, considered the two main cattle production areas of Costa Rica. No data about the BLV serological status of these farms was previously known.A transversal, descriptive, and analytical study was designed to study the BLV serological status in 379 breeding bulls belonging to 118 farms dedicated to dairy  between May 2011 and August 2018 from the coccygeus vessels using a coagulant-free vacuum tube (Vacutainer™ system). Immediately after collection, samples were kept at room temperature for 30 min to allow blood clot retraction, refrigerated at 4C°, and transported to the laboratory for centrifugation . The serum was then separated and stored at − 20 C° until analysis by an immunoenzymatic assay.At least 3 ml of blood was collected from each bull , was measured using a microplate photometer at 450 nm. With the OD obtained from each sample, the positive percentage (PP) was calculated with respect to the average of the positive control using the following formula: PP = OD of bovine serum or negative control × 100/ average OD of positive control. As recommended by the manufacturer, serum samples that yielded a PP greater than 20% were considered positive if the OD of the positive controls was greater than 1.0 and the PP of negative controls was less than 15%.The Svanovir BLV gp51-Ab from Svanova®  was used to analyze the 379 serum samples following the methodology recommended by the manufacturer. This assay reported a 100% for both sensitivity and specificity (CI 95%)  , was used as a positive control and nuclease-free water  as a negative control. The amplification steps in both rounds were 94 °C for 3 min, 40 cycles of 94 °C for 30 s, 50 °C for 45 s, 72 °C for 60 s, and a final extension at 72 °C for 5 min. Electrophoresis of all the products of the second round was performed in 1% agarose gels, using GelRed for DNA staining and carried out in an electrophoresis chamber at 100 V for 30 to 40 min. The GenRuler 100 bp DNA Ladder Plus (Fermentas®) was used as a molecular weight marker. Bands showing a molecular weight of approximately 385 bp were considered BLV-positive and sent to Macrogen Inc.  to be purified and sequenced. The partial sequences obtained were edited using the BioEdit Sequence Alignment Editor® program , using the DNeasy Blood and Tissue Kit , following the manufacturer’s recommendations. The DNA was used to amplify a segment of approximately 385 bp of the ram Hall , compareData regarding each bull, including age, breed, species, and type of livestock production system, as independent variables, were recorded with the results from the ELISA and PCR diagnostic tests (dependent variables). Fisher’s test was used to compare the percentages of positive bulls by each independent variable. Furthermore, a Kruskal–Wallis test was used to compare the bull’s age between the negatives and positives.P < 0.25 in the first step were included in the latter. A backward building model was followed based on the likelihood ratio test . At each step of the variable exclusion process, the model tested confounding and interaction by comparison of the estimated coefficients in the new model with respect to the estimated coefficients and likelihood ratio of the old model. Confounding was judged present if at least one coefficient changed more than 0.1 when the coefficient had a value between − 0.4 and 0.4, or if at least one coefficient changed more than 25% if the coefficient had a value <  − 0.4 or > 0.4 . The best-adjusted model was obtained using the Akaike information criterion (AIC).In addition, the epidemiological association between the serological condition of the bulls and the independent variables was carried out through an unconditional multivariate logistic regression using the ELISA result  as a dependent variable. The age of the bull, its species, and the type of livestock production system were included as independent variables. The logistic procedure was performed in two steps: univariate and multivariate analyses. All variables with The level of significance for all calculations was 0.05. The statistical analyses were performed in Infostat® , Jersey (n = 80), and Brown Swiss (n = 23) were the most representative breeds, while Brahman (n = 118), Nellore (n = 20), and Gyr (n = 17) were the most common Bos indicus. An overall bull BLV seroprevalence of 43.5% (165/379) was found, with an on-farm frequency of 64.4% (76/118).Of the 379 bulls tested, 169 (44.6%), 158 (41.7%), and 52 (13.7%) belonged to Bos taurus and crossbred bulls than in homologous Bos indicus . This trend was also observed in 13 out of 22 farms where both genotypes (B. taurus and B. indicus) were simultaneously present. Similarly, the proportion of seropositive bulls was higher in those from dairy and dual-purpose farms  compared to beef ranches (15.5%) (P < 0.0001). Regarding the bull’s age, the seroprevalence of BLV was higher in older than 3.5 years compared to younger sires . However, this trend was only observed in the Bos indicus.The seroprevalence of BLV was higher in Bos taurus bulls showed 3.4-fold times increased risk of being seropositive compared to homologous Bos indicus (95% CI: 1.7–6.8), while crossbreds did not differ from Bos indicus . Regarding the productive system, males belonging to dairy and dual-purpose farms showed significantly more risk of being diagnosed as positive than bulls from beef livestock (OR: 6.3 and OR: 2.9). Finally, bulls older than 3.5 years were also more likely to be BLV seropositive than younger sires. No significant risk differences for BLV were found between other age groups analyzed. All details are shown in Table  95% CI: .7–6.8, wBos taurus deposited in GenBank (AP018011 to AP18032). The sample sequence was deposited in GenBank with the accession number MN315100.Only one out of 80 semen samples from bulls yielded the amplicon of the expected size in PCR and was confirmed by sequencing. The positive sample was collected from a 42-month-old seropositive Holstein bull on a dairy farm located in the North Huetar area. BLAST results showed 100% nucleotide similarity (385/385 bp) with isolated BLV sequences from The prevalence of BLV-positive bulls (43.5%) is like previous reports in sires (41%)  and dairy cows (40%) in the country (Rodríguez Bos taurus) and crossbreds. These authors also reported in Holsteins a higher predisposition to develop persistent lymphocytosis, which may favor the virus spread up into the herd. Our significantly higher prevalence of positive BLV bulls in Holstein (75.7%) and Jersey (73.8%) compared to Bos indicus breeds  may suggest an inherent predisposition in the Bos taurus species to become BLV infected and would explain the higher frequency of positive cases found in dairy and dual-purpose farms compared to beef ones.Moreover, the frequency of BLV-positive bulls disclosed by species agrees with reports by Trainin and Brenner  and BeitBos taurus bulls had, on average, up to 35% (range 15 to 60%) of seropositivity higher than homologous Bos indicus. This picture resembles that reported by Petukhov et al. (The possible higher risk of getting BLV infection associated with the genotype is supported by the data analysis in farms that owned both species, where they were exposed to similar management practices. In those farms, v et al.  and Hassv et al. . Howeverv et al. , speciesThe lower prevalence of positive BLV cases in bulls younger than 3.5 years agrees with Radostits  and HassPrevious studies regarding the molecular diagnosis of BLV in semen from seropositive bulls have reported levels between 5.2 and 20.9% (Miller and Van Der Maaten Consequently, in seropositive sires where the genetic merit supports freezing its germplasm, the BLV DNA screening of their semen should be advised before its use in artificial insemination programs. Finally, the overall comparable prevalence of BLV seropositive bulls between the present and older studies in the country seems to indicate that the disease levels remain in cattle farms and that control decision rules must be enhanced in those systems to diminish the risk of spreading out the enzootic bovine leukosis.


The colloidal gold immunochromatographic assay (GICA) has become a popular method for the rapid detection of plant viruses. This assay format uses antibodies labeled with colloidal gold to capture and detect specific viral antigens in plant samples. GICA offers several advantages over traditional laboratory-based methods, including speed, ease of use, cost-effectiveness, and accessibility, making it an attractive option for plant virology diagnostics. Because plant viruses can cause significant economic losses in agriculture and horticulture, early detection is essential for effective management and control. Conventional laboratory-based methods such as enzyme-linked immunosorbent assays and polymerase chain reaction are sensitive and specific but require specialized laboratory equipment and training and can be time-consuming and costly. On the other hand, colloidal gold nanoparticles, specific antibodies and carefully designed components are integrated to allow visual detection of target viruses. This makes it an invaluable tool for plant disease management and monitoring that is simple and easy to perform and provides results within minutes. This review article aims to provide a comprehensive overview of the application of colloidal gold immunochromatographic assays for the rapid detection of plant viruses.  Specifications tableOne of the serious cases that highlights the importance of the identification and diagnosis of plant viruses is the globalization of trade and the increase in the human population, which will be accompanied by an increase in the demand for food across several continents of the world. Due to the lack of management and treatment methods for viral diseases, early diagnosis and detection of the pathogens demonstrate the most decisive step to stop the spread of infectious diseases, especially concerning plant viruses Several methods have been developed for plant viral diagnostics. Initially, identifying disease symptoms by eye was the simplest method to detect viral diseases. In this way, early diagnosis of the disease and the level of damage to the plants could be determined depending on the plant species and its resistance to the virus Over the last few decades, rapid and specific serological and molecular techniques have been developed for the detection of plant viruses. Serological approaches like enzyme-linked immunosorbent assays (ELISA) for virus detection, which use a specific reaction between an antibody and its corresponding antigen, are among the most important methods for diagnosing and identifying virus diseases. ELISA was first used to detect plant viruses by Clark and Adams in 1977 Researchers were encouraged by the progress of science in the field of nanotechnology to utilize antibody-antigen binding activity in order to develop highly efficient techniques based on immunosensors (The biosensors based on interactions of antigens and antibodies are known as immunosensors) According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), a biosensor is a device that can utilize a specific biochemical interaction, created by a detection component, to detect and measure a biological molecule Immunosensors are a subtype of biosensors in which the biological identifier is an antibody or antigen, and the detection process is the outcome of a specific interaction between this antibody and antigen. Typically, the antibody serves as a component of the identifier and is utilized to identify the antigen as the target species. In the design of immunosensors, like other types of biosensors, the transducers used can be optical, electrochemical, piezoelectric, or calorimetric Among the metal nanoparticles used in protein stabilization, GNPs are the most widely used. In addition to common electrostatic interactions, GNPs can stabilize proteins through covalent bonds between gold atoms and amine and cysteine groups of proteins . MethodsThe mode of action of this tool is as follows: first, the extract is prepared from the suspected sample, such as infected tissues. Approximately 100 microliters of the extract are then added to the first part. Subsequently, the extract is transferred to the second part, which contains antibodies labeled with gold particles. It is important to note that the direction of the extract flow from the first part is toward the fourth section. In the second part, if the extract contains antigens , it reacts with antibodies and gets transferred to the third part. The third part consists of two sections, namely the positive and negative control lines. In this stage, the antibodies that match the specific antigen remain in the control line, while the antibodies that do not have the corresponding antigen function as antigens for the antibodies in the control line .Fig 2A dSeveral studies have indicated that optimal pH, colloidal gold particle size, and antibody concentration are crucial factors in enhancing the efficiency of the ICA .The vast diversity of plant viruses, spanning various families, genera, and genomic structures, poses significant challenges for designing effective diagnostic assays and causes selecting appropriate antigens (proteins or nucleic acids) for diagnostic assays can be challenging. Antigens must be specific to the target virus to avoid cross-reactivity with closely related viruses. Cross-reactivity can occur when antibodies for one virus recognize similar components in another virus. This can lead to false-positive results. Some plant viruses exhibit high rates of antigenic variation, making it necessary to continuously update diagnostic reagents to keep up with the evolving virus populations. Antigen selection is a pivotal factor in achieving selectivity in GICA for the detection of plant viruses. Choosing the appropriate antigen can be done in four ways:Specific Antigens: Virus-specific antigens are specific molecular markers or structures that are distinct from a particular virus and are not found in other viruses or organisms. When these unique antigens are used in an assay, it ensures highly specific detection of the target virus and minimizes the likelihood of false positives or cross-reactivity with other pathogens Conserved Epitopes: Identifying conserved epitopes within the target virus that are less likely to be shared with other pathogens. These epitopes can be used as specific antigenic targets. Expanding research in this area may pave the way for rapid identification of viral infections. For example, common epitopes of Watermelon silver mottle virus have been identified Escherichia coli and used as an antigen to immunize mice. It was used in GICA to distinguish between these closely related tospoviruses E. coli is commonly used as a substrate for foreign gene expression E. coli and used to produce virus-specific antibodies for immunological recognition Nicotiana benthamiana. This system employs “deconstructed” viral vectors, which are delivered into plant cells via Agrobacterium-mediated systemic delivery to produce recombinant proteins. This system is also effective for generating hetero-oligomeric proteins such as immunoglobulins Recombinant Antigens: Recombinant antigens can be designed to contain only specific epitopes of interest, minimizing the risk of cross-reactivity with non-target antigens. For example, in the case of Tomato zonate spot virus (TZSV), the recombinant nucleocapsid protein of TZSV was expressed and purified in Epitope Mapping: Epitope mapping is a technique used to identify the specific regions of an antigen that are recognized by antibodies. By mapping the epitopes of target antigens, researchers can design assays that specifically detect antibodies binding to those epitopes Peptide Scanning: This involves synthesizing overlapping peptides that cover the entire antigen and testing them for reactivity with antibodies. Epitopes can be identified based on peptide binding Alanine Scanning: In this approach, specific amino acids within the antigen are mutated to alanine, and the effect on antibody binding is assessed. This helps pinpoint critical epitopes Bioinformatics Tools: Bioinformatics plays a crucial role in antigen selection and epitope mapping The selection of antibodies for the CGIA directly affects the performance of the assay Currently, one of the fastest techniques in the field of identifying plant viruses is the use of GICA Sandwich Assay Format: In a sandwich GICA, the target plant virus is captured between two specific antibodies, a capture antibody immobilized on the test line and a labeled detection antibody conjugated with colloidal gold. When the virus is present, it forms a "sandwich," resulting in a visible color change. This format is highly specific and sensitive, making it suitable for detecting plant viruses with complex structures and multiple epitopes. It minimizes the risk of false positives and can detect viruses at low concentrations. By using this format, a lateral-flow testing system has been created to swiftly identify PVX in plant leaves, offering a visual detection threshold of 3 ng/mL .Competitive Assay Format: The competitive format was designed to identify small molecules that are unsuitable for the sandwich format. In a competitive GICA, a labeled antigen competes with the target in the sample for binding to immobilized antibodies on the test strip. The intensity of the signal is inversely proportional to the sample concentration in the sample. Competitive assays are useful for detecting targets when specific antibodies are limited or unavailable . This foDirect Assay Format: In a direct GICA, antibodies labeled with colloidal gold bind directly to the viral antigen in the sample. The presence of the virus results in a visible signal on the test line. Direct assays are simple and rapid and are suitable for the detection of viruses with a single dominant antigenic site . They ar, PVY, Potato Virus M, Potato leaf roll virus (PLRV), Necrotic-type potato virus Y, Potato virus S, Potato virus A, and Clavibacter michiganensis subsp. sepedonicus. Within the immunochromatographic strip's test zone, meticulously arranged rows of spots contain antibodies tailored to target distinct potato pathogens [Multiplex Assay Format: Multiplex GICA allows the simultaneous detection of multiple plant viruses within a single assay. Different test lines or labels are used for each virus, enabling the detection of several viruses in one test. Multiplex assays are valuable for comprehensive plant virus screening, especially when multiple viruses can infect the same crop. They streamline testing and reduce the need for multiple assays. For example, a multiarray incorporated into a test strip has been designed to identify eight potato pathogens. This technology seamlessly blends the swift detection capabilities of immunochromatography with the high-throughput advantages of array techniques. These pathogens include PVXathogens .In the context of plant virus detection, selecting the appropriate GICA format depends on factors such as the specific virus being targeted, the availability of antibodies, the desired sensitivity, the complexity of the sample matrix, and the intended application. Understanding these assay formats and their suitability for different scenarios is crucial for effective plant virus diagnostics and management.One of the primary works in which gold nanoparticles have been used in the development of rapid detection methods for plant viruses is related to studies that report the use of gold-labeled antibody complexes for the detection of Brome mosaic virus (BMV), Wheat streak mosaic virus (WSMV),TMV, CPMV, PLRV, and Barley yellow dwarf virus (BYDV) –6 to 10–9 g/mL, and with the signal enhancement, the limit of detection reached up to 10–12 g/mL Methods of immunochromatographic analysis using nanoparticles of colloidal gold as markers are widely used for analytical purposes and allow visual detection of the underdetermined compound LOD can vary from one GICA to another, even for different plant viruses, due to differences in the antibodies or antigens used, assay conditions, and the format of the assay. For example, the increased affinity of the anti-PPV compared with primary antibodies was investigated and studied several times, and finally, this test was appropriate for the virus . Also, tThe LOD can be improved by using some techniques. For example, a straightforward method was devised to enhance the sensitivity of the sandwich lateral flow immunoassay by modifying the binding conditions between a polyvalent antigen and gold nanoparticle-antibody conjugates. In this investigation, the polyvalent antigen employed was the PVY, while the antibodies were coupled with gold nanoparticles measuring 17.4 ± 1.0 nm in diameter. The dried GNP conjugates were applied on the test strip both before and after pre-incubation with the sample. Interestingly, a 30-second pre-incubation of the GNP conjugates and the sample led to a remarkable 60-fold improvement in the detection limit, reducing it from 330 ng∙mL−1 to just 5.4 ng∙mL−1 compared to the conventional lateral flow immunoassay. The quantitative outcomes of this enhanced lateral flow immunoassay, involving pre-incubation, were substantiated via ELISA, yielding a robust correlation coefficient of 0.891 Rapid Results: One of the primary advantages of colloidal gold immunochromatographic assays is their speed. They provide quick results within minutes, enabling rapid screening and detection of plant viruses User-Friendly: These assays are designed to be user-friendly and do not require specialized laboratory equipment or technical expertise. They are simple to use and can be performed in the field, allowing for on-site testing without the need for complex laboratory infrastructure. Methods like PCR and sequencing require specialized equipment and trained personnel, whereas GICA can be used by non-specialists . For exaPortability: Colloidal gold immunochromatographic assays are portable and can be easily carried to different locations. This mobility is advantageous for surveillance purposes, as it allows for efficient monitoring of plant virus outbreaks across various fields or regions Visual Interpretation: The results of these assays are typically interpreted visually, often as colored lines or signals Citrus ringspot virus, or 'Candidatus Liberibacter asiaticus'. Furthermore, the ICS test successfully identified CYVCV in tissue extracts at a 1:320 (w/v) dilution, matching the efficiency of the dot-ELISA assay. In summary, the ICS assay offers a cost-effective, faster, and simpler alternative to conventional CYVCV detection methods, making it a valuable tool for large-scale CYVCV detection or monitoring efforts Sensitivity and Specificity: Colloidal gold immunochromatographic assays have been developed to provide high sensitivity and specificity in the detection of viruses. They utilize antibodies or other specific binding agents that recognize viral antigens, ensuring accurate identification of target viruses while minimizing false positives or negatives . The ICSCost-Effectiveness: Compared to more complex laboratory-based techniques, colloidal gold immunochromatographic assays are generally more cost-effective. They offer a relatively inexpensive solution for rapid screening and surveillance, making them accessible to a wider range of users, including farmers, agronomists, and plant health inspectors. PCR and sequencing can be expensive due to the cost of reagents and specialized equipment, making GICA a more budget-friendly option for routine monitoring , Tobacco Vein Banding Mosaic Virus (TVBMV), and PVY. This test boasts limits of detection, reaching 200 ppb (ng/ml) for TMV, 1 ppm (µg/ml) for PVY, and 2 ppm for TVBMV particles Multiplexing Capability: GICA can be adapted for multiplexing, enabling simultaneous detection of multiple plant viruses in a single test, which is essential for comprehensive screening. Multiplexing with PCR can be complex and requires careful primer design and optimization. A Triplex Lateral Flow Immunoassay has been created for the swift diagnosis of three viruses: TMVIt's worth noting that while colloidal gold immunochromatographic assays have numerous advantages, they also have limitations.Sensitivity: Colloidal gold immunochromatographic assays may have lower sensitivity compared to other more sophisticated laboratory techniques like PCR or ELISA. This means that the assay may not be able to detect low levels of the virus, leading to false-negative results Specificity: The assay's specificity, or the ability to accurately detect the target virus without cross-reacting with other substances, can be a concern. Depending on the design and quality of the antibodies used, there is a possibility of false-positive or false-negative results due to non-specific binding or cross-reactivity with similar viruses or substances Detection Limit: The detection limit of colloidal gold immunochromatographic assays may not be as low as other molecular-based methods. This can limit their usefulness in detecting viruses present at very low concentrations . So, GICCross-Reactivity: Some GICA assays may exhibit cross-reactivity with closely related viruses or substances, potentially leading to misinterpretation of results, but another method such as PCR can be highly specific when properly designed, reducing the risk of cross-reactivity. To evaluate the efficacy of GICAs, one of the tests required is a cross-reactivity test. All tests performed after demonstrating that there is no cross-reactivity with closely related viruses of the target virus will be publicly presented and made available .The colloidal gold immunochromatographic strip assay has emerged as a powerful and efficient tool for the rapid detection of plant viruses. This review has provided a comprehensive overview of the various aspects associated with this assay, including its principles, components, advantages, limitations, and applications. Importantly, the colloidal gold immunochromatographic strip assay has demonstrated significant potential for on-site testing in field conditions, enabling early detection and rapid response to plant virus outbreaks. Its simplicity and cost-effectiveness make it particularly suitable for resource-limited settings, where sophisticated laboratory facilities may be lacking. While the assay has proven successful in detecting numerous plant viruses, further research and development are necessary to address certain challenges, such as enhancing the assay's sensitivity for low titer infections and improving its specificity to minimize cross-reactivity. Additionally, the integration of emerging technologies, such as smartphone-based detection and digital image analysis, holds promise for further enhancing the assay's convenience and accuracy. Overall, the colloidal gold immunochromatographic strip assay is a valuable and versatile tool for rapid plant virus detection. Its potential applications in plant disease management, quarantine measures, and crop protection make it an essential technique for safeguarding agricultural productivity and ensuring food security. Continued research and innovation in this field will undoubtedly contribute to the development of even more advanced and efficient diagnostic tools, ultimately benefiting the agricultural industry and global food production.Not applicable.Abozar Ghorbani: Supervision, Conceptualization, Methodology, Data curation, Writing – review & editing, Visualization, Investigation. Sajad Astaraki: Data curation, Writing – original draft. Mahsa Rostami: Data curation, Writing – review & editing. Arezoo Pakdel: Data curation, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We aimed to understand whether ankle dorsiflexion range of motion (ROM) and dynamic knee valgus (DKV) kinematic inter-limb asymmetries would be associated with the Lateral Step-Down Test (LSD) in basketball players with chronic ankle instability (CAI), patellofemoral pain (PFP) and healthy controls (HC). An observational cross-sectional study with a between-subject design was employed. Female basketball athletes with CAI (n = 20), PFP (n = 20) and HC (n = 20) were recruited. Ankle dorsiflexion-ROM, DKV angle during a single-limb squat, and LSD quality were measured bilaterally. The Asymmetry index (ASI) was calculated to identify between-limb percentage imbalances. The correlation matrix between the tasks was calculated. Ankle dorsiflexion-ROM was less in the CAI and PFP than in the HC group regardless of limb (p < 0.001). DKV angle was greater in the CAI and PFP than in the HC group bilaterally (p < 0.001). LSDs were similar between the PFP and CAI groups (p = 0.698) but worse than the HC group (p = 0.001). The ASI showed asymmetry across all tasks (p < 0.001), with the greatest asymmetry for the DKV angle. The correlation matrix between tasks on both limbs was significant (p < 0.05). Our findings suggest significant asymmetries in ankle dorsiflexion-ROM and frontal plane knee control are present in female basketball athletes with CAI and PFP, and thus, highlights need to evaluate and reduce limb asymmetries in these populations. Musculoskeletal injuries, particularly of the lower extremity, cause short-term disability, interfere with participation in physical activity, and are associated with joint disorders in later life2. Thus, the general goal of preventing acute and chronic musculoskeletal injuries and reducing the associated burden among high-risk individuals is of considerable interest. Many studies report a higher prevalence of musculoskeletal pain among females in the general population3. When scholastic and colleagiate athletes are considered, females continue to experience greater rates of injury, particularly to the knee and ankle experiencing higher prevalences of Patellofemoral pain (PFP) and chronic ankle instability (CAI) in weight-bearing sports  compared to their male counterparts4. PFP is a multifactorial clinical condition resulting from abnormal patellofemoral joint loading, increasing joint stress and producing retropatellar pain5. Conversely, CAI represents both mechanical and functional instability of the ankle joint resulting in repetitive bouts of instability following an initial lateral ankle sprain injury6. PFP and CAI represent two of the most prevalent musculoskeletal injuries among physically active individuals4 and, while these conditions affect different joints, a commonality among them is that patients with these musculoskeletal disorders experience aberrant movement patterns that may contribute to/arise from the disorder’s presence7.Many health benefits accrue from consistent physical activity across the lifespan. Participation in physical activities, however, carries an inherent risk for acute and chronic musculoskeletal injury7. Though few researchers have investigated DKV angle in persons with CAI, those who have studied it found DKV angle to be greater in this population compared to their health counterparts9. Accordingly, an increased DKV angle is not only associated with painful knee conditions but can also be related to dysfunctions in other lower limb joints (e.g. the ankle)10. Reduced ankle dorsiflexion range of motion (ROM) has been reported as a risk factor for some conditions, such as patellar tendinopathy11, Achilles tendinopathy13, chronic ankle instability14, metatarsal stress fractures11, and anterior knee pain15.Previous studies have reported that dynamic knee valgus (DKV), an aberrant movement pattern during weight-bearing activities, is a significant risk factor in patients with PFP14. Therefore, restriction of ankle dorsiflexion ROM represents a possible risk factor for excessive DKV angle17 during squat and jump landing tasks18 and is linked to injurious landing mechanics19. With restricted ankle dorsiflexion ROM, individuals may try to compensate with movement in the frontal or transverse plane throughout the overall kinetic chain17, thus, creating an increased DKV angle20. This compensation may present as foot pronation20, internal tibial rotation20, hip internal rotation and adduction21, pelvic drop15, or gastrocnemius and soleus tightness22. Patients with PFP22 and CAI14 commonly display a limited ankle dorsiflexion ROM with adverse functional consequences for the knee joint biomechanics during landing9, ascending/descending stairs, squatting, jumping and running23. Restricted dorsiflexion will also increase impact forces / mechanical loads and redistribute them proximally to the knee and hip in weight-bearing activities such as the Lateral Step-Down test (LSD)14. The LSD is a simple, clinician-friendly tool designed to assess lower extremity movement quality during a functional activity24. The test allows clinicians to identify faulty movement patterns and evaluate the trunk, hip and knee behaviour during the task24. Since its creation, the LSD has been used to assess patients’ quality of movement with CAI and PFP15. There are several advantages of using LSD to assess movement quality, mainly because it is a quick and easy test to perform in a clinical environment24. Moreover, the performance of the LSD score is influenced by a reduced ankle dorsiflexion ROM in individuals with PFP and CAI15. Therefore, ankle dorsiflexion ROM represents a key assessment when patients demonstrate a lower movement quality during an LSD15.In a closed kinetic chain, the ankle acts as a firm base of support such that its movement restriction and/or instability can affect the function of proximal joints25. Neuromuscular asymmetry of the lower limbs is associated with potential injury and can be used to predict future injury or re-injury26. Inter-limb asymmetries (ILA) may potentially place both legs at an increased risk of injury in sports; the strong leg may sustain excessive stress due to high dependence and loading (i.e. overuse), whereas the weak leg may be compromised to a potentially lower load tolerance capacity27. In addition, ILA have been associated with an increased risk of sports injury because the asymmetries may result in unequal force attenuation or a loss of frontal plane stability, which are important to sustain the impacting forces26. For example, research has examined lower ILA, mainly in healthy basketball players, during functional movements 28, leaving uncertainty about their role in athletes with CAI and PFP. Individuals with PFP may have gluteus medius muscle activation asymmetry, which may be associated with pain severity29. Also, Nakagawa et al.30 showed that male military recruits with greater asymmetry on the Y-Balance Test (YBT) posterolateral direction and Frontal Plane Knee Projection Angle (FPKPA) during single-leg squat were at a greater risk of developing PFP30. However, Plastaras et al.31 showed early stages of unilateral PFP in female runners was not associated with hip abduction strength asymmetry. Regarding patients with CAI, the asymmetry between right and left anterior reach distances (> 4 cm) and limitations in posterolateral reach distances  represent risk factors for lateral ankle sprains and may contribute to CAI32. Tajdini et al. showed that patients with CAI walk with greater ILA in vertical ground reaction force and muscle activity across the gait cycle compared to the non-CAI group33.Quantifying neuromuscular control between legs (e.g. reduced function or performance in one limb compared to the other) is critical to identify individuals potentially at risk of injury, establishing when an athlete can return to play following injury, and optimizing strength and conditioning training14, to our best knowledge, there is a lack of research concomitantly investigating all three tasks  in both lower limbs  among basketball players presenting PFP and CAI. From a clinical perspective, a better understanding of these phenomena would inform researchers and clinicians  to decide on the assessment, treatment evolution, and improving movement control of these patients considering both limbs and functional relationship of the lower kinetic chain34.Although limited dorsiflexion ROM and its effect on excessive DKV and lower movement quality have been reported in PFP and CAITherefore, this study aims to understand whether dorsiflexion ROM and DKV kinematic ILA would be associated with the LSD scores in basketball players with CAI or PFP compared to healthy controls, and whether differences exist in these measures between populations. We hypothesized that: (1) there would be significant differences in DKV angle, ankle dorsiflexion ROM, and LSD scores in CAI and PFP compared to healthy controls; (2) there would be no significant difference between CAI and PFP in DKV angle, ankle dorsiflexion ROM, and LSD scores; (3) ankle dorsiflexion ROM and DKV asymmetry would be associated with LSD scores asymmetry in PFP and CAI; and (4) there would be an inverse association between ankle dorsiflexion ROM and LSD scores and a direct relation between DKV angle and LSD scores.This observational cross-sectional study was conducted and reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. The study conformed to the Declaration of Helsinki. All participants provided written informed consent. The ethics board of the Sport Science Research Institute (IR.SSRC.REC.1400.120) approved this study.35. A physiotherapist with 13 years of experience in diagnosing and treating patients with musculoskeletal disorders assessed all the athletes, classifying them eligible when presenting with unilateral CAI, unilateral PFP or healthy conditions based on the inclusion and exclusion criteria reported in Table Participants in this study were recreational, female, basketball athletes recruited from sports clubs in Iran. Participants were between 20 and 30 years of age . The population of female basketball players were chosen due to the high prevalence of CAI and PFPG-Power software  was used to estimate the sample size according to a recent similar study that assessed DKV angle and LSD. The analysis revealed that to perform repeated measures, between factors Multivariate Analysis of Variance (MANOVA) with an effect size of 0.30, power of 0.30, and alpha of < 0.05, we would need at least 19 participants. To account for potential dropout, we determined a minimum sample size of 60 participants was required to be assigned into three groups: 20 participants with CAI, 20 with PFP, and 20 healthy females.36 and the Persian version of Foot and Ankle Ability Measure (FAAM) for activities of daily living (ADL) and SPORTS subscales37.The CAI group completed a series of patient-reported outcomes surveys to confirm a history of significant ankle sprains/giving way/recurrent ankle sprains, including the Iranian version of the Cumberland Ankle Instability Tool Questionnaire (CAIT)38 and the Clark test39.The PFP group were assessed by the Numerical Pain Rating Scale (NPRS)36, FAAM-ADL and FAAM-SPORTS37, NPRS38 and Clark test39.The Healthy group completed the CAITIn the pre-screening phase, the research group distributed recruitment flyers by hand (brochures) to basketball clubs’ meetings in the metropolitan area of Tehran. Before participating in the study, all athletes were briefed about the objectives and read and signed the informed consent form. Then, the included participants attended a baseline assessment as follows:Next, participants reported their age and involved limb, had their height (cm) and body mass (kg) measured using a tape measure and a digital scale, respectively, and their body mass index (BMI) calculated.Moreover, the ankle dorsiflexion ROM and DKV angle were measured, and the LSD was performed for both lower limbs. The first limb measured (right or left) was randomized, but ankle dorsiflexion ROM measures were always obtained before the DKV angle and LSD assessments. All the assessments were conducted with the participants barefoot at Kharazmi University in January 2020. An examiner with 13 years of experience that received 5 h of training in ankle dorsiflexion ROM, DKV angle, and LSD assessments performed all the measurements. The examiner was blinded to the group allocation to avoid bias during the evaluation.40. Ankle dorsiflexion ROM in the weight-bearing lunge (WBLT) was measured with an Acumar™ digital inclinometer . The participant put the tested foot along a 50-cm-long line drawn on the ground, and a continuous 60-cm-long line was drawn on a wall. A test taker placed an inclinometer on the anterior aspect of the tibia, 15 cm distal to the tibial tuberosity, so that the line bisected the heel, and the second toe was on the line. The subject lunged forward and directed their patella as close as the line drawn on the wall while the heel remained in contact with the floor. This puts the ankle joint in maximal dorsiflexion. Once maximal dorsiflexion was reached, the ankle dorsiflexion ROM was recorded , representing a valuable assessment strategy41. In this study, the tests were conducted for the intraclass reliability of the examiner, resulting in a correlation coefficient (within-session reliability) of 0.95 (0.92–0.97).A digital inclinometer was used to record weight-bearing ankle dorsiflexion ROM by measuring the tibia angle to the ground during a two-point staggered upright position8. Participants were asked to stand on the test limb, facing the video camera and then to squat down at an angle of at least 45° knee flexion but not greater than 60°, for 5 s. The knee flexion angle was checked during practice trials (maximum of three) using a standard goniometer observed by the same examiner throughout all trials42. Trials were only accepted if the participant squatted within the desired range of knee flexion and maintained their balance while keeping their hands on their iliac crests throughout the trial42. Two-dimensional videos of the single-leg squat were captured using a Canon Vixia HF R42 digital camera (Canon USA), sampling at 60 frames per second. The camera was placed at the height of the subject’s knee, 3 m anterior to the participant’s squatting area, and aligned perpendicular to the frontal plane. DKV angle was calculated as the angle formed between the thigh and shank segments . Within-day ICCs showed good reliability and ranged from 0.59 to 0.88, and between-days ICCs were good to excellent, ranging from 0.72 to 0.9142. In this study, the tests were conducted for the intraclass reliability of the examiner, resulting in a correlation coefficient (within-session reliability) of approximately DKV 0.92 (0.90–0.95).We measured the DKV angle during a single-leg squat. The single-leg squat was chosen because it allows for easy visualization of poor neuromuscular control (e.g. increased DKV angle) in patients with PFP and CAInts Fig. b. Specif24. Participants stood in a unilateral stance atop a 20-cm step with the medial border of their foot near the stair edge and their hands on the hips. The limb not being tested was held off the step over the floor. In the next stage, they were asked to slowly descend the stair by flexing the test limb knee so that the contralateral heel touched the ground and returned to the initial position. This maneuver was repeated for 5 repetitions. Participants received no feedback on performing the task and no information on performance errors. A video camera  aligned with the frontal plane was used to record the assessments for subsequent scoring , and inability to sustain unilateral balance (e.g. bearing weight given the opposite limb)15. If the DKV angle exceeded the foot midline, an extra point was given for a total of 6 points. Higher scores (more errors) indicated poorer movement quality 15. Pilot data from our lab suggest inter-rater agreement on the LSD to be substantial (kappa = 0.74), which aligns with previously established values in the literature (k = 0.67- 0.81)43.The LSD measured the quality of functional movementsing Fig. c. There 44 ASI =  leg- involved (or right) leg/contralateral leg) × 100.The Asymmetry index (ASI) was calculated to identify functional imbalances between limbs using the following formula:45 as (SD/mean) × 100 for each participant and then averaged across all participants.To confirm that inter-limb differences are meaningful, we calculated the coefficient of variation (CV)We considered the right side as the involved leg for the healthy group when calculating the ASI.t tests were adopted to further elucidate differences between the involved and contralateral legs in three tasks . For non-parametric data (LSD), the Kruskal Wallis and Mann–Whitney U tests were used to determine the difference between groups. To determine the association between tasks on the involved and contralateral limbs, the relationship between ankle dorsiflexion ROM measures, DKV angle, and the LSD score was determined using a Spearman rank correlation coefficient. Correlation values were interpreted as follows:  < 0.25 little or no relationship, 0.25–0.50 fair relationship, 0.50–0.75 moderate to good relationship,  > 0.75 good to excellent relationship46. The alpha value was set at p < 0.05 for all analyses. Finally, effect sizes (ES) (bias-corrected Hedges’ g) and 95% confidence interval (CI) were also computed to estimate the precision and magnitude of group differences, given the multiple t tests ran. Effect sizes were interpreted as trivial (≤ 0.19), small (0.20–0.59), moderate (0.60–1.19), large (1.20–1.99), very large (2.00–3.99), and extremely large (≥ 4.00) for paired and independent comparisons47.The independent variables in this study were group  and limb . Dependent variables were LSD, DKV angle, and ankle dorsiflexion ROM. The SPSS software  was used to analyze the data collected in this study. Descriptive statistics were adopted to summarize the data with measures of central tendency. The Shapiro–Wilk and also Kolmogorov–Smirnov tests assessed the normal distribution of the tasks. A Two-way mixed model ANOVA was used to compare the interaction of group on limb on each dependent variable. The Scheffe test was used for multiple comparisons for the post hoc test. Paired The study was approved by Research Ethics Committees of Iran institute of sport science . This study was performed in accordance with the standards of ethics outlined in the Declaration of Helsinki.One hundred and twenty-nine basketball players were screened, 76 participants were assessed for eligibility based on the inclusion/exclusion criteria. Next, we selected 24 participants from the PFP group, 26 from the CAI group, and 26 from the healthy group. Finally, considering the dropout and based on the exclusion criteria, we included 60 participants (20 participants in each group). The flowchart of the participants’ selection is reported in Fig. There were no significant differences between groups in age, height, mass, and BMI  in each group separately Table . The greThe relation between all tasks on both the involved and contralateral sides was significant Table . Further30.This study aimed to quantify differences in ankle dorsiflexion ROM, DKV angle, and LSD across groups as well as determine the association between limb and ankle dorsiflexion ROM, DKV angles, and the LSD ILAs scores in basketball players with CAI, PFP and healthy controls. We observed that female basketball players with PFP and CAI have impaired ankle dorsiflexion ROM, greater DKV angle, poorer scores in the LSD, and greater ILAs than healthy controls Tables a–c. Diff14, who reported that patients with CAI had reduced ankle dorsiflexion ROM compared to healthy subjects14. Similarly, in the study by Rabin et al.15 movement pattern was assessed visually during an LSD and rated as "good" or "moderate in PFP patients15. They demonstrated that ankle dorsiflexion ROM was more limited among participants with a moderate versus a good quality of movement15. Importantly, our ankle dorsiflexion ROM values exceeded the previously established minimum detectable difference of 4° and 6°, suggesting these differences may impact patient function41.The ankle dorsiflexion ROM in the involved limb was 9° less in the CAI group and 6° less in the PFP group compared to the healthy control group Table , Fig. 3a48. Accordingly, the mean difference observed in patients with CAI and PFP of over 8° between groups is clinically relevant and in agreement with the results of previous investigators who demonstrated greater DKV angle in females with than without PFP49. Although many studies emphasize that DKV angle is a significant risk factor in patients with PFP7, to our best knowledge, only one study8 has confirmed that patients with CAI demonstrated increased DKV angle compared with coper and control subjects8. The paucity of studies investigating DKV angle in CAI offers future research opportunities. Additionally, these results collectively suggest the need to evaluate and improve ankle dorsiflexion ROM as well as DKV angle in patients with both PFP and CAI.The DKV angle in the single-leg squat test revealed that the involved limb angle was nearly 8° greater in the CAI group and approximately 9° greater in the PFP group compared to healthy controls Table , Fig. 3b50. There is, however, limited research investigating LSD in women with CAI. In one study that was conducted, the authors reported individuals with poor movement quality had significantly less ankle dorsiflexion ROM compared to the good movement quality group14.In terms of LSD results, our study revealed that CAI and PFP performed, on average, moderate quality movement 3.45), while healthy groups performed on average good quality movement (0.55)  compared to the PFP group (40°) Table , Fig. 3a53. Moreover, a study examining the performance of patients with PFP during the single-leg squat and Y-balance test showed results consistent with our findings30. They showed that male military recruits with greater asymmetry on the Y-Balance test posterolateral direction, and frontal plane knee projection angle were at a greater risk of developing PFP30. This asymmetry may suggest a neuromuscular imbalance between limbs. Side-to-side imbalances in neuromuscular strength, flexibility, and coordination represent significant predictors of increased injury risk54. Asymmetry between two limbs is considered a risk factor in athletes28. In the present study, comparing athletes with PFP and CAI, it seems that athletes with PFP experience more asymmetry and motor deficits on the contralateral side. Therefore, including the entire motor chain on both lower limbs is significant in evaluating and designing prevention and treatment programs. In addition, further prospective studies are needed to explore whether there is a cause-effect relationship between inter-limb asymmetry and injury development.Our asymmetry analyses revealed a significant association between ankle dorsiflexion ROM and DKV with the LSD scores in female basketball athletes with PFP and CAI in support of our third hypothesis Tables , 5. Our 28. Therefore, the DKV is likely a critical task for detecting ILA between legs. In addition, prospective research is needed to determine if there is a direct correlation between DKV angle asymmetry and injury rate over time. Moreover, it is challenging to establish an ASI threshold for typical values on the other tasks examined in this research because of the scarcity of research on this topic.In addition, we aimed to determine which task has the greatest sensitivity to identify asymmetries based on ASI calculation. ASI varied among ankle dorsiflexion ROM, DKV angle, and LSD tasks and indicated that the percentage of asymmetry could differ depending on the task performed. Accordingly, this finding indicates that obtaining measurements on various independent tasks is important to assess ASI. The largest ASI detected in our study was found in DKV for CAI and PFP. This ASI is higher than the 10–15% threshold describing the potential risk of injury among participants as reported in the literature40. During the LSD, maximal ankle dorsiflexion ROM is required to allow the heel to remain in contact with the ground14. A limitation in ankle dorsiflexion ROM or the proximal lower limb joints may require compensation from other joints16. Decreased ankle dorsiflexion ROM will limit the forward progression of the tibia over the talus, resulting in compensatory subtalar pronation, which, in turn, may displace the knee medially55. Previous studies suggest that limited ankle dorsiflexion ROM may be associated with a greater medial knee displacement during various functional activities18; such compensation may increase DKV angle. Less research has been conducted to investigate the influence of limited ankle dorsiflexion ROM on a frontal and transverse plane lower limb alignment. Bell et al. found that ankle ROM and strength rather than hip ROM and strength were associated with DKV angle during a controlled squat56. In the above study, the DKV angle was corrected by adding a heel lift, confirming the role of limited ankle dorsiflexion ROM in dynamic knee valgus during a loading task56. Bell-Jenje et al.21 demonstrated that participants with restricted dorsiflexion ROM had greater dynamic knee valgus during the LSD21. Also, they showed that the association between an increase in hip adduction ROM and decreased dorsiflexion ROM during a functional loading task such as the LSD test emphasizes the importance of the kinetic chain in lower limb alignment21. In the closed kinetic chain, the relative angle, magnitude, and direction of one motion-dependent body segment affect another segment, leading to differences in how these segments interact21. This finding is significant in a clinical context where limited dorsiflexion ROM is associated with CAI14, PFP15, and dorsiflexion ROM may, therefore, play a key role in predicting injuries21, but further longitudinal research is needed. Additionally, these results collectively suggest the need to evaluate and improve ankle dorsiflexion ROM to improve movement quality in patients with both PFP and CAI. There are few studies investigating this relationship in CAI. So, future research is needed to investigate a relationship between CAI and other lower limb injuries during functional tasks ; possible use of posture software for the movement analysis is further suggested. Also, a future prospective study is needed to examine whether increased DKV angle with reduced dorsiflexion ROM may contribute to a greater risk of knee injury in patients with CAI.The fourth hypothesis’s results demonstrated a moderate to good relationship between ankle dorsiflexion ROM and LSD scores and a good to strong relationship between DKV angle and LSD scores. These findings agree with similar studies investigating ankle dorsiflexion ROM and LSD scores in CAI and PFP57. Accordingly, prospective studies should consider the cause-effect relationship between inter-limb asymmetry and injury development. Secondly, we have recruited only a sample of female non-professional basketball players with CAI and PFP. Thus, our findings could not be generalized to male participants of different ages, professional athletes, players of other contact sports or in the presence of different clinical conditions (e.g. ACL reconstruction). Thirdly, the small effect size and power used in sample size estimation is another limitation that should be considered in future research. Fourthly, this study did not examine hormone profiles and status or inquire about contraception usage, which can have important implications for physiological function in female participants58. Lastly, we assessed the quality of movement only using the LSD and adopted a 2D analysis of limb motion. Therefore, future studies should consider investigating a motor task similar to basketball-specific movement techniques  and also integrating a 3D analysis  combined with a real-time electromyography data acquisition to gain further insight into the importance of ankle dorsiflexion ROM and DKV angle during sport-specific tasks.This study presents some limitations that should be acknowledged. Firstly, we cannot obtain causative conclusions from our study by adopting a cross-sectional designFemale basketball players with PFP and CAI have impaired ankle dorsiflexion ROM, greater DKV angles, poorer scores in the LSD, and greater ILAs compared to healthy controls. Ankle dorsiflexion ROM and DKV were associated with LSD. These findings inform clinicians and researchers of the need to evaluate ROM and function throughout the kinetic chain and in both lower limbs in injured athletes tailoring specific rehabilitation programs.