Bacterial infection and poor cell recruitment are among the main factors that prolong wound healing. To address this, a strategy is required that can prevent infection while promoting tissue repair. Here, we have created a silver nanoparticle-based hydrogel composite that is antibacterial and provides nutrients for cell growth, while filling cavities of various geometries in wounds that are difficult to reach with other dressings. Silver nanoparticles (AgNPs) were synthesized by chemical reduction and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and inductively coupled plasma-mass spectroscopy (ICP-MS). Using varying concentrations of AgNPs , several collagen-based silver–hydrogel nanocomposite candidates were generated. The impact of these candidates on wound healing was assessed in a rat splinted wound model, while their ability to prevent wound infection from a contaminated surface was assessed using a rat subcutaneous infection model. Biocompatibility was assessed using the standard MTT assay and in vivo histological analyses. Synthesized AgNPs were spherical and stable, and while hydrogel alone did not have any antibacterial effect, AgNP–hydrogel composites showed significant antibacterial activity both in vitro and in vivo. Wound healing was found to be accelerated with AgNP–hydrogel composite treatment, and no negative effects were observed compared to the control group. The formulations were non-cytotoxic and did not differ significantly in hematological and biochemical factors from the control group in the in vivo study. By presenting promising antibacterial and wound healing activities, silver–hydrogel nanocomposite offers a safe therapeutic option that can be used as a functional scaffold for an acceleration of wound healing. Chronic wounds affect millions of patients all around the world and contribute significantly to their morbidity and mortality ,2,3. AmoHydrogels are promising candidates for wound dressings due to their extraordinary properties such as biocompatibility, tunable mechanical properties, and good permeability . The hig+ ions at a greater rate than bulk silver, by virtue of their large specific surface area [A previous body of work published by our group describes the synthesis and complete characterization of a novel hydrogel-based scaffold shown to be biocompatible and result in improved healing of open wounds in a mouse wound healing model ,13,14. Gace area ,20. In tBacterial infection and poor cell recruitment are two primary factors that can significantly impede the process of wound healing in patients with chronic ulcers. As a result, finding innovative approaches that effectively address these issues and simultaneously facilitate endogenous tissue repair through structural scaffolding holds immense promise. Such a novel strategy would not only prevent bacterial infection within the wound but also promote the natural healing process, leading to expedited and improved wound healing outcomes. Previously, we developed a thermosensitive collagen–GAG-based hydrogel that is liquid at low temperatures (<4 °C), solidifies within 10 to 20 min of application to the wound sites, and contains all the amino acids, vitamins, and minerals required for cell growth. Being in a liquid form when applied to the wound, it fills up all the cavities and void areas seen in many different skin defects, injuries, and non-healing wounds. To improve the current product such that it would be able to manage infection and protect wounds from bacterial contamination, we used AgNPs to add antibacterial activity to the hydrogel. For this purpose, first we synthesized and characterized AgNPs. Next, lyophilized collagen–GAG hydrogel powder was reconstituted in AgNPs colloidal solution with different concentrations of AgNPs up to 600 ppm to form Ag–hydrogel nanocomposite.AgNPs were synthesized by chemical reduction method and then characterized. The synthetic pathway involved using two new reducing agents that are safe and non-toxic, which allowed for the synthesis of Ag nanoparticles with narrow and unimodal size distribution.Physicochemical properties of AgNPs are key factors in antibacterial activity and toxicity. We used dynamic light scattering (DLS) and transmission electron microscopy (TEM) to measure size, dispersity, and zeta potential of nanoparticles. The TEM images showed nanoparticles had a spherical shape with an average diameter of 23.34 ± 4.37 nm a,b. DLS These results were further confirmed by UV–Vis spectroscopy, which is the most widely used technique for structural characterization of AgNPs. The optical properties of spherical AgNPs are highly dependent on nanoparticle diameter and change when particles aggregate. The absorption spectrum of AgNPs solution immediately after synthesis displayed a symmetrical strong absorption peak, associated with the Surface Plasmon Resonance (SPR), at 410 nm. This finding is consistent with the previous studies, showing that the SPR peak for the spherical AgNPs normally appears between 410 and 480 nm. In addition, the absence of any additional peak and the symmetrical shape of the plasmon band were a good indicator of its monodispersity. As for stability assessment over time, the same characteristic absorption band was detected during 6-month storage at 4 °C c, displa50 value of 64 ppm  and Pseudomonas aeruginosa (PA), which are the most common bacterial species responsible for wound infections. The possible mechanism of the AgNPs’ antibacterial activity is attributable to a combined effect of both AgNPs and Ag ions including attachment and disruption of the bacterial membrane, damage of intracellular biomolecules and structures, and the induction of oxidative stress with generation of reactive oxygen species (ROS) and free radicals [AgNPs have broad-spectrum antibacterial activity against different bacterial species including Methicillin-resistant radicals .MRSA and PA were found to be 79.18 µg/mL and 6.29 ± 0.690 µg/mL, respectively. These may be different from values reported in the literature, mainly due to differences in the specific bacterial strains used in a given study. In addition, a direct comparison is not possible as characteristics specific to the AgNP used in a given study that impact activity may differ, including variation in the particle’s physicochemical properties such as size, shape, crystallinity, surface chemistry, and capping agent [S. aureus, while that for PA was closer to those reported earlier [S. aureus are more resistant to AgNPs compared to Gram-negative bacteria such as P. aeruginosa, which may be the result of differences in the overall composition between these bacterial groups, resulting in differences in overall thickness and outer membrane surface charges [In our study, the MICs of AgNPs against ng agent . While s earlier ,32,33. S charges . For ins charges .MRSA and PA. As shown in PA compared to only 1.71 ± 0.35 for the 100 ppm sample. Similarly, the log reduction against MRSA was 4.56 ± 0.26 for the 600 ppm sample compared to 2.04 ± 0.69 for the 100 ppm sample.Given that the AgNPs need to retain their activity when incorporated into the actual hydrogel, we verified the antibacterial activity of the Ag–hydrogel nanocomposite against MRSA (106 CFU/mL) prior to suturing the implantation site to induce an infection  was found with Ag–hydrogel containing 600 ppm Ag that completely prevented the colonization of the underlying Ti discs with MRSA.Next, we assessed the potential for the hydrogel to prevent the colonization of a substrate surface that is encased by the hydrogel itself as this most closely mimics the scenario where a hydrogel-treated surface may be challenged with a higher load of contamination bacteria. For this, we cast Ti discs inside hydrogel or Ag–hydrogel and exposed them to a suspension containing 10PA, AgNP–hydrogel composite showed better antibacterial activity against MRSA in the penetration test. This could be because PA is capable of two distinct types of surface-specific motilities, twitching and swarming [Interestingly, while the MIC of AgNPs was lower for swarming .These results confirm that in addition to high cytocompatibility, the AgNP–hydrogel composite exhibited strong antibacterial activity. Such behavior has been reported previously in different synthetic and natural polymers including PVA, PVP, gelatin, and alginate used to prepare antimicrobial Ag–hydrogel nanosystems. Travan et al. described synthesis and stabilization of AgNPs in a chitosan-derived polysaccharide solution and studied the cytotoxicity and antibacterial properties in both solution and within a 3D hydrogel structure. Antimicrobial results showed that the nanocomposite system displays a very effective bactericidal activity toward both Gram-positive and Gram-negative bacteria. However, the hydrogel does not show any cytotoxic effect towards the three different eukaryotic cell lines including mouse fibroblast-like (NIH-3T3) cells. They suggested that the nanoparticles, immobilized in the hydrogel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while entrapping AgNPs in hydrogel could prevent them from diffusing into the surrounding environment and making them available for cells to be taken up .Chronic non-healing wounds have a significant impact on numerous patients every year, significantly contributing to their morbidity and mortality. Complications and delayed wound healing are often a result of bacterial infection. Thus, wound dressings with advanced antibacterial activity are of great interest within wound care management. Antibiotic-loaded dressing with controlled release activity can provide antibacterial activity while avoiding the exposure of the healing tissue to toxic concentrations of active agent .To assess the effect of Ag–hydrogel on the wound healing process, we utilized the rodent excisional wound healing model. Considering that the rodent skin is mobile, wound closure for the most part takes place via contraction. To overcome this shortcoming, we used an excisional splinting model in rats. In this model, a splinting ring is tightly attached to the skin around the wound to prevent skin contraction, forcing the wound to heal through granulation and re-epithelialization, a process similar to that occurring in humans.As shown in In addition to antibacterial activity, AgNPs play an active role in wound healing. Previous studies have shown that AgNPs promote the migration of fibroblasts and stimulate the differentiation of fibroblasts to myoblasts, which speed up the wound contraction and promote the healing process . AgNPs aIn this manuscript, we discuss the synthesis and characterization of collagen-based Ag–hydrogel nanocomposites with excellent wound healing and antibacterial properties. The findings of this study address some of the major shortcomings of currently available wound healing products including bacterial biofilm formation, infection development, and resultant impaired wound healing. The incorporation of AgNPs into an already highly effective hydrogel scaffold resulted in significant antibacterial and pro-wound healing activities including accelerated re-epithelization and better wound healing without cytotoxic effects. Given this, the Ag–hydrogel nanocomposite developed herein has significant potential to be a safe therapeutic option as a functional scaffold for the acceleration of wound healing.3) as a source of silver was reduced by trisodium citrate and glucose as reducing agents. A 1 mL volume of 30 mM trisodium citrate was added to 100 mL of distilled deionized water (DDW) under constant stirring and heated up. As the temperature reached 95 °C, 2 mL of 5 mM AgNO3 and 50 mM glucose were added to the solution drop by drop. The reaction mixture was stirred vigorously on a magnetic stirrer and heated until the solution color turned light yellow. The resulting AgNPs solution was kept in dark at 4 °C for future use. The solution was further centrifuged  and resuspended in DDW to prepare aqueous AgNPs with different concentrations. To form Ag–hydrogel nanocomposite, lyophilized collagen–GAG hydrogel powder was reconstituted in AgNPs aqueous solution with different concentrations.Silver nanoparticles (AgNPs) were synthesized by chemical reduction method ,43. SilvSynthesized AgNPs were visualized using transmission electron microscopy . AgNPs solution was diluted with DDW. Then, a drop of the solution was placed on a TEM grid and dried at room temperature. The AgNPs were viewed at an operating voltage of 80 kV, and the diameter of 100 particles was measured using ImagJ to assess the particle size and distribution. The particle size distribution of AgNPs was also confirmed using dynamic light scattering  before proceeding to Ag–hydrogel nanocomposite preparation. The AgNPs solution was diluted with DDW and transferred to a square plastic cuvette. Solid-state laser (35 mW red diode) with a wavelength of 640 nm was used, and the scattering detection angle was chosen to be 90° . Another4 cells/well with a pre-warmed fresh medium  and incubated at 37 °C in an incubator supplied with 5% CO2 for 24 h. After that, the cells were treated with different concentrations of silver nanoparticles  and incubated for another 24 h. Next, the test solutions were removed, 100 μL of MTT-containing medium (1 mg/mL) was added to each well and further incubated for 4 h. Then, the supernatants were removed and replaced with 100 μL DMSO to solubilize the resulting purple formazan crystals formed in living cells. The relative cell viability (%) was calculated based on the absorbance at 570 nm measured with a microplate reader  according to the following formula:The cytotoxic effect of synthesized silver nanoparticles was measured using MTT assay. Human dermal fibroblasts (HDF) cells were seeded in a 96-well tissue culture plate at a density of 1.5 × 104 cells per well of a 96-well plate with a DMEM-high glucose supplemented with 10% FBS and a 1% penicillin/streptomycin solution and incubated at 37 °C and supplied with 5% CO2. After 24 h of incubation, the culture medium was removed, each well was washed with phosphate-buffered saline (PBS) three times, and then 100 µL of extracts was added per well. Cell proliferation and viability was determined using MTT assay after 24 h of exposure.The cytocompatibility of Ag–hydrogel nanocomposite containing different concentrations of AgNPs  was evaluated based on the International Organization for Standardization 109993-5 using extract analysis. In brief, formulations were extracted in culture medium at 37 °C for 24 h, and the extracts were collected for cell culture. Human dermal fibroblasts (HDF) cells were seeded at a density of 1.5 × 105–106 of each bacterial strain were inoculated with the serially diluted AgNPs in 96-well plates and incubated for 24 h. After that, viable counting colonies were enumerated using serial dilution of a 10 µL aliquot from each sample in PBS followed by growth on agar Petri dish by incubating for 14–18 h at 37 °C, according to the following equation:A standard broth microdilution method was used to determine the minimal inhibitory concentrations (MIC) of synthesized AgNPs against American type culture collection (ATCC) strains of Methicillin-resistant Staphylococcus aureus (MRSA-USA300) (Gram-positive) and Pseudomonas aeruginosa (PAO1-#166.lux) (Gram-negative). In brief, a total of eight serially diluted samples were used, starting with a 200 ppm stock solution of AgNPs, two-fold diluted in ddH2O  seven additional times. The bacterial isolates were cultured and sub-cultured in LB as the growth media under aerobic conditions at 37 °C, 48 and 24 h prior to all experiments. A starting colony-forming units (CFU) of 10MRSA and PA. Briefly, 200 µL/well of hydrogel was added to 48–well plate and kept at room temperature for 10–15 min to cast. Then, microbial culture containing 106 CFU/mL of bacteria was added to the well and then placed in 37 °C incubator. After 24 h, small aliquots of the bacterial solution were removed and microorganisms in it were enumerated as stated above.Two different methods were applied to evaluate the antibacterial activity of the formulations in vitro. In the first method, the disc-shaped casted hydrogels were directly placed in bacterial solution to be in direct contact with two common wound infecting bacteria, In the second method, the effect of Ag–hydrogel nanocomposites in preventing bacteria penetration into the hydrogel was evaluated. To do so, a sterile disc-shaped Titanium (Ti) wire was placed inside the AgNP–hydrogels prior to casting. Similar to previous method, the hydrogel was placed in bacterial solution after casting. The Ti disc was subsequently harvested, and number of bacteria infiltrated into the hydrogel and attached to the Ti disc was counted after 24 h incubation at 37 °C.Healthy female Sprague–Dawley (SD) rats  were obtained from Charles River Laboratories. Animal care and all experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the University of British Columbia Animal Care Committee. The clearance to conduct this study was provided by the University of British Columbia Committee of Animals and Ethics . The rats were maintained with water and standard laboratory chow ad libitum for 1 week prior to the experiment to acclimatize to the laboratory environment in a temperature-controlled room. The animals were randomly divided into study groups and were individually anesthetized using 2% isoflurane in oxygen (flow rate of 1.0 L/min) and kept under anesthesia throughout the procedure.6 MRSA inside each pocket. Following implantation, the incisions were closed with absorbable sutures. On day 4 post-surgical implant, animals were sacrificed and the whole pockets and surrounding tissue were harvested. Harvested tissues were then homogenized using a Precellys 24 tissue homogenizer, and the number of bacteria were quantified by serial dilution in PBS, followed by plating on agar and CFU counting in 14–18 h.To determine the efficacy of Ag–hydrogel nanocomposite formulations on bacterial growth inhibition in an in vivo setting, we used a subcutaneous infection wound model that was recently used for evaluation of antibacterial activity of different biomaterials using rats . After fThe rat excisional wound splinting model was used to determine the efficacy of Ag–hydrogel nanocomposite in wound healing. Following aseptic surgical techniques, two full-thickness excisional wounds with 8 mm circular diameter were created bilaterally on either side of the vertebral column of each rat. To overcome the problem of wound contracture in rodent wounds, silicone splints with an inner and outer diameter of 10 and 14 mm were secured on the edges of the wound. Five experimental groups were tested with either: (1) clean untreated wounds or (2) clean wounds filled with CG hydrogel alone; (3–5) clean wounds filled with Ag–hydrogel nanocomposite containing 200, 400, and 600 ppm of Ag, respectively. The wound healing process was monitored every 3 days for up to 3 weeks.® software , Bethesda, MD, USA) for surface area changes. Total reduction in the wound size was calculated by dividing the surface area at each time point to that of the original wound using the following equation:Digital images of the wound area were captured on different time points. The images were processed with ImageJAt the end point of the study, all wound sites were harvested for histological evaluation. Tissue samples were fixed in 10% neutral buffered formalin solution  for 24 h before being processed and embedded in paraffin blocks. Samples were sectioned at 5 µm thickness and stained using Masson’s Trichrome stain. Briefly, slides were incubated in Bouin’s solution for 1 h at 60 °C. Then, rinsing slides were stained with Weigert’s Hematoxylin solution (Sigma-Aldrich) followed by Biebrich Scarlet-Acid Fuchsin, phosphomolybdic–phosphotungstic acid, aniline blue, and 1% acetic acid solution. Collagen appears blue, nuclei black, and cytoplasm red.To examine the safety and evidence of systemic inflammatory response, blood cell count and biochemical assay were conducted at the end of animal study. Blood samples were obtained by cardiac puncture. Separate portions of blood were transferred to tubes containing either lithium heparin or trisodium EDTA as anticoagulants. The whole blood was used to count total red blood cells (RBC), white blood cells (WBC), platelets, and hemoglobin (or hematocrit). The blood serum was processed in a VETSCAN Analyzer  for the analysis of the following parameters: total protein (TP), alkaline phosphatase (ALP), alanine transaminase (ALT), glucose (GLC), blood urea nitrogen (BUN), and creatinine (CR).t-test and one-way or two-way analysis of variance (ANOVA) with GraphPad Prism 9.0.0 (121) . Post hoc test was performed using the Dunnett test. Mean values were considered to be statistically significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).All data were presented as the mean ± standard deviation of three independent experiments. Data were subjected to statistical analysis using paired Student’s


The application of biochar and zeolite either alone or in combination with mycorrhiza increased significantly soil pH and EC compared to mycorrhiza treatment and untreated soil. It can be concluded that the combination of biochar and mycorrhizal inoculation has great potential as a cost-effective and environmentally technique for enhancing heavy metal immobilization, lowering heavy metal availability and plant uptake, and improving cowpea plant growth.Heavy metal pollution of agricultural soil has become a major serious concern. The development of suitable control and remediation strategies for heavy metal contaminated soil has become critical. The outdoor pot experiment was conducted to investigate the effect of biochar, zeolite, and mycorrhiza on the bioavailability reduction of heavy metals and its subsequent effects on soil properties and bioaccumulation in plants as well as the growth of cowpea grown in highly polluted soil. Zeolite, biochar, mycorrhiza, zeolite with mycorrhiza, biochar with mycorrhiza, and soil without any modifications were the six treatments used. The experiment was conducted with a completely randomized design and four replications. The results indicated that the combination of biochar with mycorrhiza had the highest values of root and shoot dry weight and the lowest heavy metal concentrations in root and shoot as well as bioconcentration and translocation factors for all heavy metals. The highest significant reductions in the availability of heavy metals over the control were found with biochar with mycorrhiza Heavy metal accumulation was higher in vegetable soils than in other agricultural soils2. These metals have detrimental consequences on soil fertility, plant development and yield, and food safety. Therefore, finding effective remediation approaches for lowering heavy metal bioavailability in the soil resulting in lower plant uptake and phytotoxicity of heavy metals, which led to improved plant growth is critical. The safe and cost-efficient approach to remediate heavy metals in soil is the use of stabilizing agents. Some soil amendments such as biochar and zeolite and its effect on heavy metals have been identified as cost-effective methods for the immobilization of toxic metals4.Heavy metal pollution of agricultural soils has been recorded in many regions throughout the world. The main sources of heavy metals in agricultural soil are the excessive application of sewage sludge, agrochemicals products, wastewater and organic manure5. It's been proven that biochar improves soil fertility and properties as well as helps to stabilize heavy metals6, thereby decreasing the phytoavailability of heavy metals through electrostatic attraction, physical adsorption, ion exchange, co-precipitation, and complexation7. Moreover, biochar enhances nitrogen fixation by improving the symbiotic performance of legumes with rhizobia8. Zeolites are crystalline and alkaline earth metals or hydrated aluminosilicate alkali metals with negatively charged positions and high specific surface area that can efficiently remove metals9. Zeolites have a great potential for remediation of heavy metal-contaminated soil by producing holes for water retention and heavy metal adsorption10, and due to their high cation-exchange capacity and hence capability to absorb heavy metals and so limit their phytoavailability11.Biochar is a carbon-rich substance produced from organic feedstocks at high temperatures (> 300 °C) under oxygen-limited conditions. Biochar is a very porous substance with a large specific surface area12. Mycorrhiza plays a vital role in plant growth by enabling nutrient gain from soils. Furthermore, mycorrhiza plays a significant role in the remediation of contaminated soils. The inoculation of mycorrhiza in contaminated soils reduces the negative effects of heavy metals. It has been shown to be able to survive toxic metals and to act as a filtration barrier to reduce heavy metals from the transformation in the soil–plant system, affecting the uptake and accumulation of heavy metals in plants and decreasing their toxicity13. Several studies have shown that arbuscular mycorrhizal fungi (AMF) reduces heavy metal toxicity in a variety of plants, and to maximize mycorrhiza's efficacy in toxic metal reduction, a variety of amendments have shown promising results in combination with mycorrhiza such as biochar14 and zeolite15.The use of large-surface-area amendments in heavy metal-contaminated soil improves plant growth, which can be linked to these components' involvement in lowering heavy metal availability in the soil and, as a result, encouraging soil microbial activityVigna ungiculata L.) is one of the legume crops valued for its high nutritional value, which provides protein, minerals, and vitamins for both humans and animals. It is an essential part of sustainable agriculture. It can fix nitrogen (N) from the atmosphere and grow well on infertile soils. However, cowpea is among the crops that readily absorb heavy metals, which can cause major health problems in humans16. Excessive accumulation of heavy metals in the soil results in a gradual decrease in plant growth, photosynthetic activity and production17. Many synthetic and natural amendments can decrease heavy metal extractability from contaminated soils. However, they are unable to increase microbial activity, soil productivity and plant growth. Soil treatment with mycorrhiza, biochar, and zeolite is considered an environmentally beneficial and cost-effective remediation technique. However, there is a scarcity of current study findings on the interaction of mycorrhiza with biochar or zeolite in soil remediation for vegetable production. Therefore, the objective of this study was to evaluate the effect of mycorrhiza and soil ameliorants (biochar and zeolite) and their mixture on soil characteristics, transformation and bioaccumulation of heavy metals in plants, and the availability of heavy metals in the soil as well as the growth of cowpea grown on heavily polluted soil.Cowpea  > Cr (10.88 mg kg−1) > Pb (5.93 mg kg−1) > Zn (3.79 mg kg−1) > Ni (3.26 mg kg−1) > Cd (1.32 mg kg−1) > Co (0.62 mg kg−1). The values arranged in decreasing order as follows: control > mycorrhiza > zeolite > biochar > zeolite + mycorrhiza > biochar + mycorrhiza. The highest significant reductions over the control were found with biochar + mycorrhiza, which were 59.1%, 44.3%, 38.0%, 69.7%, 77.8%, 77.2% and 73.6% for Cd, Co, Cr, Cu, Ni, Pb and Zn, respectively.The results showed that there were significant effects of different applications on the content of available heavy metals in the soil after harvest Fig. . The con–1 for soil pH and EC, respectively.The application of biochar and zeolite either alone or in combination with mycorrhiza increased significantly soil pH compared to mycorrhiza treatment and untreated soil Fig. , which t6. Nawab et al.18 found that the root and shoot growth of pea plants decreased with the high toxicity of Cr, Ni, Zn, Pb, and Cd in soil. The present study found that mycorrhiza can boost shoot biomass and adding biochar and zeolite to the soil can promote it  were used19. The increases in dry biomass as a result of biochar and zeolite treatment can be explained by increased nutrient availability and uptake in the soil and/or plant23 and the improvement in soil physio-chemical properties24.Biochar and zeolite boost plant growth during the remediation process by a variety of mechanisms, including toxic metal adsorption, higher soil water retention, encouraging plant growth-promoting microorganisms, and increased root development25 reported that biochar improved nodule formation in mungbean plants, microbial activity in the soil, and increased shoot fresh biomass. Increased mycorrhizal colonization in the soil causes an increase in plant biomass26. Mycorrhiza help plants absorb water and nutrients by forming a pervasive hyphal network, increasing the efficiency of chemical fertilizers27. In metal-contaminated soils, mycorrhiza has been found in association with plant roots and has been suggested to protect plants against heavy metal toxicity and improve plant development28. Plants grown in heavy metal-contaminated soils can benefit from mycorrhiza by facilitating nutrient uptake, protecting them from metal toxicity, sequestering HMs into mycorrhiza structures, and improving phytostabilization 23 On the other hand, using biochar in combination with other amendments, such as soil microorganisms, can boost biochar's trace metal remediation capacity and improve crop growth29. The stimulative effect of biochar and mycorrhiza combined treatment could be attributed to the benefits of biochar to mycorrhiza as a source of reduced carbon compounds and available nutrients 23.Moreover, Bashir et al.31. Besides, biochar absorbs the substances that are hazardous to mycorrhizal fungus, effectively lowering the toxicity of many heavy metals32. Guo and Li32 found that the combination of biochar and mycorrhiza protected the plants from heavy metal toxicity and promoted plant growth and the effect of their combined inoculation were greater than a single one.Biochar amendment increases the physicochemical properties of soils, making them more suitable for mycorrhiza colonization by promoting spore germination, hyphal branching, and mycorrhiza growth 26. Mycorrhiza's efficiency to reduce Cd uptake increased after zeolite addition to the soil15. The application of biochar with mycorrhiza in heavy metal-contaminated soils reduced significantly heavy metal levels in the shoots by reducing heavy metal translocation from the roots32.The heavy metal concentrations in roots and shoots of the plants grown under biochar or zeolite combined with mycorrhiza inoculation applications were lower than in single applications, which were related to the interaction of biochar or zeolite with mycorrhiza. These significant reductions could be attributed to enhanced immobilization of available heavy metals in soil Fig. , as well33. Metals adsorb on the surfaces of biochar and mycorrhiza, reducing their concentrations and mobilization in the soil, as well as their translocation in plants35. Moreover, biochar increase the pH, organic carbon content, and cation exchange capacity of the soil leading to lower the phytoavailability of heavy metals37.The bioaccumulation factor results indicate that all treatments of soil amendments and mycorrhiza inhibited the bioaccumulation of selected heavy metals in cowpea Table . The acc38. Moreover, mycorrhiza is effective in reducing heavy metal transport from the root to the shoots26, because heavy metals can be retained in mycorrhizal structures39. Plant-mycorrhiza symbiosis could enable selective transport mechanisms of both essential minerals and heavy metals40. Mixing biochar with mycorrhiza in metal-contaminated soils reduced significantly heavy metal translocation from the roots32.The decrease in TF values Table  could be41, resulting in a reduction in the plant's heavy metal uptake42. Both zeolite and biochar supply alkalinity to the soil causing insoluble metals 43. In this study, the pH value of soil is increased with the application of biochar and zeolite  supplying alkalinity and consequent pH rise43 (2) promoting sorption by surface complexation45, (3) increasing cation exchange retention19, (4) organic complexation47 , and (5) reduction of hydraulic conductivity and pore size of soils19. Due to metal binding and complex formation, zeolite was effective in lowering heavy metal mobility46. The positive cations in zeolite can exchange with specific heavy metal cations in soil solutions, giving it a high capacity for cation exchange48. Biochar can absorb soil heavy metals by ion exchange process, complexation, precipitation functions, electrostatic attraction, or transforming metals from an inorganic form to an organic form that altered the pollutants' bioavailability and mobility7. On the other hand, mycorrhizal soil inoculation could be vital in immobilizing heavy metals in the soil and altering their availability to plants28.The increase in soil pH and organic matter will promote heavy metal immobilization28. On the surface of the fungal structure, positive charge particles such as amino acids, cysteine, glutathione and thiol groups adsorb and later reduce heavy metals from one form to another35. Also, mycorrhiza creates extracellular polymeric substances, which have carboxyl, phosphoric, amine and hydroxyl functional groups and have a high affinity for metal adsorption through chelation, surface precipitation, and ion exchange mechanisms49. The fungal structure is often much finer than roots, and it has a strong potential to chelate heavy metals and reduce their bioavailability in the rhizosphere through metal speciation51. Mycorrhiza decreases heavy metal toxicity in plants by keeping heavy metals in mycorrhizal structures such as the fungal mycelium and vesicles, where high quantities of heavy metals are concentrated, preventing their mobilization to aerial plant tissues27. Moreover, many studies have shown that combining mycorrhiza and biochar has a positive impact on lowering bioavailable heavy metals in soil52.Mycorrhiza acts as a physical barrier by sequestering the heavy metals into mycorrhizal structures and immobilizing heavy metals in plant roots43. The increase in soil pH after the application of biochar alone or in combination with mycorrhiza may be related to the pH of biochar itself , at a depth of 20 cm. A standard approach narrated in Ryan et al.The zeolite clinoptilolite which a hydrated aluminosilicate mineral that contain alkali and alkaline earth metals  was used in this experiment. It was purchased from a company in Giza, Egypt. The zeolite was sieved at 0.2 mm before being used. The biochar was produced using dried woods taken from mature trees of mango as feedstocks. They were exposed to direct sunshine before being sliced into little 10 cm pieces. Dry wood chips were covered thoroughly with aluminum foil to provide an oxygen-limited condition and pyrolyzed at 500 °C for 5 h in muffle furnace . Biochar was cooled to room temperature before being crushed using a stainless-steel mill and sieved with a 2 mm mesh size. Mycorrhiza fungi used in this experiment were obtained from Soil, Water and Environment Research Institute, ARC, Egypt. The mycorrhiza used is an endomycorrhizal biofertilizer with 250 spores in 1 g of the carrier. The properties of zeolite and biochar used in this study are shown in Table The outdoor pot experiment was conducted at Mansoura Horticulture Research Station, Dakahlia Governorate, Egypt, during the summer season of 2021. The experiment was conducted with a completely randomized design in four replications. The following six treatments were applied: zeolite, biochar, mycorrhiza, mixtures of zeolite and mycorrhiza, mixtures of biochar and mycorrhiza, and control (soil without any modifications). Both biochar and zeolite were applied at a rate of 2% w/w and completely mixed with soil alone to obtain homogeneity.4.8H2O, CoCl2.6H2O, Cr2(SO4)3, CuSO45H2O, Ni-SO46H2O, Pb(C2H3O2)2 and ZnSO47H2O) at a concentration of 3.2, 10, 85, 50, 50, 70 and 130 mg kg-1 for Cd, Co, Cr, Cu, Ni, Pb and Zn, respectively. The water solution with toxic metals was thoroughly mixed into the soil to ensure homogeneity then the plastic pots with a capacity of 7 kg were filled with contaminated soil. The pots were left to equilibrate for three weeks before seed sowing. During this time, the moisture content of the soil was maintained at 65 percent of its field capacity.To prepare the artificially polluted soil, all of the pots were contaminated with a solution of seven heavy metal salts  content, and electrical conductivity (EC). The elemental content of C (carbon) was determined using the Thermo Scientific Flash 2000 elemental analyzer. The total and available concentrations of Cd, Co, Cr, Cu, Ni, Pb and Zn in the extracts were determined using Thermo Scientific TM ICAPTM 7000 Plus Series ICP-OES Ammann57. The available heavy metals were extracted by 0.005 M DTPA at pH 7 according to Lindsay and Norvel58.For particle-size analysis, the hydrometer method was used. Jackson's method59, to evaluate the heavy metal absorption and translocation. The ratio of metal concentration in plant tissues to metal concentration in the soil is known as BCF, while the ratio of metal concentration in the plant shoot to metal concentration in the plant root is known as TF.The bioconcentration factor (BCF) and translocation factor (TF) of each heavy metal were determined according to Padmavathiamma and Li60. The Least Significant Difference (LSD) test was used to evaluate the differences between means. The significance of the difference was determined using the P = 0.05 value.The ANOVA technique was used to calculate statistical analyses by Costat computer software according to Snedecor and CochranThis study was developed with commercial seeds, therefore nonexotic or at risk of extinction, under controlled conditions, meeting all institutional, national and international guidelines and legislation for cultivated plants.


Diabetic kidney disease (DKD) is a severe diabetic complication that affects up to half of the individuals with diabetes. Elevated blood glucose levels are a key underlying cause of DKD, but DKD is a complex multifactorial disease, which takes years to develop. Family studies have shown that inherited factors also contribute to the risk of the disease. During the last decade, genome-wide association studies (GWASs) have emerged as a powerful tool to identify genetic risk factors for DKD. In recent years, the GWASs have acquired larger number of participants, leading to increased statistical power to detect more genetic risk factors. In addition, whole-exome and whole-genome sequencing studies are emerging, aiming to identify rare genetic risk factors for DKD, as well as epigenome-wide association studies, investigating DNA methylation in relation to DKD. This article aims to review the identified genetic and epigenetic risk factors for DKD. Urinary albumin excretion can be classified as normal or mildly increased, moderately, or severely increased albuminuria; the two latter ones are also called micro- and macroalbuminuria. The classical view has been that albuminuria represents an earlier sign of DKD, followed by reduced eGFR and eventually kidney failure, but a substantial proportion of individuals with DKD may present with reduced kidney function even without albuminuria . On the ysiology . Lack ofDKD is a complex multifactorial disease in which both genetic and environmental risk factors contribute to the development and progression of the disease. However, the exact molecular mechanisms leading to DKD remain poorly understood. Apart from albuminuria and eGFR, no other biomarkers are yet in clinical use for monitoring disease progression or identification of individuals at risk, and only a few treatment options exist for the prevention of DKD, especially in individuals with T1D. To address these issues, genetic studies aim to identify the underlying molecular mechanisms leading to DKD. Here, we review the genetic factors that have been identified for DKD, mainly based on genome-wide association studies (GWASs) performed within the latest decade and summarize the main findings from epigenetic studies—being the potential dynamic link between genes and the environment—investigating the DNA methylation changes associated with DKD.2Three decades ago, family studies reported clustering of DKD in siblings with T1D, suggesting an inherited component of the disease –17. More33.1AGTR1 on chromosome 3q (ADIPOQ (NCK1 (CNDP1 gene associated with both DKD and serum carnosinase concentrations (The early genetic studies on DKD utilized various microsatellite markers and single-nucleotide polymorphisms (SNPs) for family-based linkage studies to identify chromosomal regions co-segregating with DKD. One of the strongest linkage peaks with a logarithm of odds (LOD) score of 3.1 was obtained in a candidate gene study of the osome 3q , and manosome 3q –31. Subs  transporter  has bee56094641  in the h4LDLR mutation carriers had hypercholesterolemia in the UK Biobank WES data , the resolution remains low even in the GWAS-based linkage studies, hindering further fine-mapping and interpretation of the results.While common variants have a large effect on complex traits at the population level , the lowWES data . To idenWES data . Many ofclerosis  and FRASsyndrome . OverlapD in T2D . While aβ dehydrogenase 14 (HSD17B14) gene exome-wide significantly  associated with the disease progression from DKD to kidney failure. The gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models , and a KRT6B splice-site variant rs425827 associated with DKD . Both variants were associated with DKD also when compared with diabetes controls without DKD, but with lower statistical significance . Furthermore, gene aggregate analyses identified ERAP2  and NPEPPS ; both are expressed in the kidney and implicated in the renin-angiotensin-aldosterone system–modulated immune response  and protein tyrosine kinase 2 (PTK2) involved in DKD  study of 76 Finnish sibling pairs with T1D but discordant for DKD found significant enrichment of variants in DKD in gene promoter and enhancer regions, as well as for specific transcription factor binding sites , but lard in DKD ; of noteteinuria . A recenteinuria , and lar51c-associated sites explained as much as 71 to 97% of the association between HbA1c and diabetic complications in the DCCT  that can induce changes in gene expression without changing the underlying sequence. In contrast to an individual’s genetic variation, which is constant across tissues and throughout lifetime, epigenetic modifications are dynamic and modifiable. Thus, epigenetic changes may vary between tissues, cell types, and developmental stages and can even be affected by environmental factors. Furthermore, in disease states, the methylation patterns can change either as a cause or a consequence of the disease . In thisthe DCCT , furtherDNA methylation is the most frequently studied epigenetic modification and occurs at cytosine bases of cytosine–phosphate–guanine dinucleotide sites (CpGs) in the DNA sequence. In addition to DNA methylation, additional epigenetic modifications exist, such as histone modifications (acetylation and methylation), and their role in DKD has also been explored. For example, dysregulation of histone H3 lysine 27 trimethylation (H3K27me3) in TGF-β1–induced gene expression has been associated with DKD . Histone5.1p-value below 9 × 10−8 has been suggested as a threshold for robust significance, adequately controlling for the false positive rate for the EPIC array , in studies including both T1D and T2D (UNC13B gene. An intronic SNP (rs2281999) in the same UNC13B gene was identified for DKD in T1D in a prior genetic association study including genetic variants in 127 candidate genes . The most recent and largest study, including 1,304 individuals with T1D, identified 32 sites with altered methylation in DKD , in addition to age, sex, and six cell-type proportions. These seven included two intergenic CpGs on chromosome 19 and four CpGs located within genes PTBP3, NME7, SLC1A5, and SLC27A3 and one CpG within a long non-coding RNA (LINC01800).To date, methylation levels at over 150 CpG sites across the genome have been associated with DKD, eGFR, or albuminuria  and SLC27A3 (cg21961721) gene, both with higher methylation levels in DKD in T1D  in the general population , none of the differentially methylated CpG sites has emerged in subsequent EWASs.Although most of the DNA methylation association studies performed on DKD have covered the whole genome, targeted approaches have been undertaken as well. Swan et al. evaluated DNA methylation levels associated with DKD for CpGs located within genes influencing mitochondrial function in 442 individuals with long term T1D . AlthougTXNIP gene has been recurrently observed in the context of diabetes and glycemia, such as persistently higher HbA1c both in T2D and T1D   found an overlap with DKD risk factors including dyslipidemia , HbA1c (TXNIP), blood pressure  and obesity   improved prediction of eGFR decline even when baseline eGFR and Albumin-to-creatinine ratio (ACR) were included in the model  or cg17944885 (between ZNF788P and ZNF625-ZNF20), suggesting that methylation changes observed at these sites are consequential to kidney disease or its other manifestations, e.g., hyperglycemia. Kim et al. used Mendelian randomization in the opposite direction, i.e., to assess the causal effect of metabolic phenotypes on CpG methylation changes identified in their EWAS on T2D (n = 8) and DKD (n = 3). These analyses revealed that fasting glucose resulted in 2% hypomethylation of cg00574958 located in the CPT1A gene, whereas HbA1c or BMI did not causally affect the cg00574958 methylation. Genetically determined eGFR, however, was associated with 7% hypomethylation of cg19693031 within TXNIP , as well as hypomethylation of all the CpGs identified for DKD in T2D, including three CpGs within genes: COMMD1, TMOD1, and FHOD1.Because of the dynamic nature of epigenetic changes, the methylation changes observed at CpGs in DKD can be either a cause or a consequence of the disease. To separate the causal methylation changes from the consequential, EWASs have also attempted Mendelian randomization, which uses genetic information to infer causality , 128. AlD in T1D . On the 6During the last 5 years, both GWAS and EWAS have identified an expanding number of genetic loci for DKD. Nearly 80 genetic loci have reached genome-wide statistical significance for DKD, albuminuria, or eGFR in diabetes to date. Much of this increase is not only due to larger meta-analyses of existing diabetes cohorts but also due to CKD studies in the general population including a substantial number of individuals with diabetes, as well as general population biobank studies. Even larger meta-analyses combining multiple biobank studies are likely to result in more genetic loci contributing to DKD. One of the major challenges of such studies will be how to best ascertain cases with DKD, either based on ICD codes that do not capture DKD well, self-reported DKD, or single measurements of albuminuria or eGFR, both of which vary over time. General population biobanks may also be affected by selection bias including healthier than average individuals , leadingDM = −0.019, betanoDM = −0.011, Pdiff = 1.3 × 10−27; TPPP, rs4663171, betaDM = −0.011, betanoDM = −0.004; Pdiff = 2.5 × 10−9), potentially reflecting the elevated risk and accumulated risk factors for kidney complications among individuals with diabetes. On the other hand, genetic risk factors for DKD in T1D seem to differ from the general population  that may be used for risk stratification and identification of affected traits and phenotypes. In general population, PRS on eGFR was associated with incident CKD and kidney failure in the Atherosclerosis Risk in Communities study with 8.6% of the individuals having diabetes . In diabTo date, several CpG sites with altered methylation levels in DKD have been identified across the genome. Understanding the underlying mechanism behind these changes would be critical, i.e., are the observed changes driven by kidney disease or some other manifestation that emerges as the disease progress, and whether the changes are causal for the development or progression of DKD. In addition, methylation levels are also influenced by the genetics. Insights to the complex network behind the findings might therefore require integrating DNA methylation results with results from multiple other sources such as GWAS as well as transcriptomic and proteomic data. Some efforts in that direction have already been made. Indeed, a recent study demonstrated that DNA methylation explains a larger fraction of kidney disease heritability than gene expression by integrating GWAS data with methylomic and transcriptomic data obtained from 446 kidney tissue samples .DNA methylation markers have proven useful for the prediction of DKD progression. Current studies, however, have focused on the later stages of kidney disease, when AER is severely increased or when kidney failure has occurred. EWASs at earlier stages of DKD, when AER is only moderately increased, could potentially identify additional CpGs and perhaps even more importantly, enable the prediction of early changes using DNA methylation. Although DNA methylation scores have not yet been as extensively implemented in risk prediction as the PRSs, methylation scores show a great promise as they incorporate information from both the genes and the environment. In a recent study, methylation scores improved the prediction of a range of clinical diagnoses and traits, including kidney disease, outperforming the predictive ability of polygenetic risk scores . HoweverNS and ED revised the literature and wrote the manuscript. P-HG critically revised the manuscript for the scientific content. All authors agree to be accountable for the content of the work. All authors contributed to the article and approved the submitted version.


Sao Tome & Principe (STP) has a high peri-neonatal mortality rate and access to high-quality care before childbirth has been described as one of the most effective means of reducing it. The country has a gap in the coverage-content of antenatal care (ANC) services that must be addressed to better allocate resources to ultimately improve maternal and neonatal health. Therefore, this study aimed to identify the determinants for adequate ANC utilization considering the number and timing of ANC contacts and screening completion.P-value < 0.05.A hospital based cross-sectional study was undertaken among women admitted for delivery at Hospital Dr. Ayres de Menezes (HAM). Data were abstracted from ANC pregnancy cards and from a structured face-to-face interviewer-administered questionnaire. ANC utilization was classified as partial vs adequate. Adequate ANC utilization was defined as having ANC 4 or more contacts, first trimester enrolment plus one or more hemoglobin tests, urine, and ultrasound. The collected data were entered into QuickTapSurvey and exported to SPSS version 25 for analysis. Multivariable logistic regression was used to identify determinants of adequate ANC utilization at p = 0.005] and age above 35  when comparing with women aged 14–19 years, urban residence , and planned pregnancy  were the determinants of adequate ANC utilization.A total of 445 mothers were included with a mean age of 26.6 ± 7.1, an adequate ANC utilization was identified in 213  and a partial ANC utilization in 232 . Age 20–34 [AOR 2.27 (95% CI: 1.28–4.04), Less than half of the pregnant women had adequate ANC utilization. Maternal age, residence and type of pregnancy planning were the determinants for adequate ANC utilization. Stakeholders should focus on raising awareness of the importance of ANC screening and engaging more vulnerable women in earlier utilization of family planning services and choosing a pregnancy plan, as a key strategy to improve neonatal health outcomes in STP. Poor antenatal healthcare (ANC) utilization is an international public health challenge especially in low-resource countries, with prejudice toward the health of the expectant mother and the unborn child , 2. AcceAntenatal care status is typically characterized only by the number or timing of visits or ANC contacts . HoweverEvidence-based screenings are also endorsed by the WHO, including a core set of interventions such as blood pressure measurement, iron tablet supplementation, tetanus toxoid vaccination, urine testing, hemoglobin test, and rapid tests for sexually transmitted diseases . These sNonetheless, there is a gap in the actual use and implementation of these ANC screening practices, mainly in sub-Saharan African (SSA) countries .The magnitude of the gap varies, with greater concerns reported in relation to underuse compared to overuse, mainly in screening completion .Factors linked to maternal ANC utilization, are usually grouped into community-level features, sociodemographic and pregnancy-related characteristics, media exposure and availability of maternal health services . EffectiSao Tome & Principe (STP), a sub-Sharan Island country in the Gulf of Guinea, is characterized by a total fertility rate of 4.4 (number of births per woman) and a considerable unmet need for contraception of 33.7% . AlthougThe country already reached two out of four 90/90/80/80 targets and milestones for maternal and newborn health, with 95.4% women receiving the service of a skilled birth attendant at birth and 91% newborns obtaining first postnatal care within the first 24 h after birth . MissingMost studies about ANC in Sub-Saharan countries dichotomize the outcome variable ANC into utilized and not utilized—only based on the total number of visits/contacts—disregarding whether the WHO´s recommended screenings were completed , 13. MisAn institutional-based cross-sectional analytical study was conducted from July 2016 to November 2018 in the only country´s hospital, Hospital Dr. Ayres de Menezes (HAM).2, 859 km2 of which for Sao Tome and 142 km2 for Principe with approximately 200.000 inhabitants [The archipelago of STP has two islands, with a total land surface of approximately 1,001 kmabitants . All sixabitants . Completabitants , 9, 16. This study was conducted in HAM which has a maternity unit responsible for 82,4% of all deliveries in the country , 10. An All pregnant women admitted to the HAM maternity unit for delivery constituted the source population, whereas the study populations were selected pregnant women admitted to the HAM maternity unit during the study period.The eligibility criteria for participants were as follows: 1) all women admitted to the hospital for delivery with a gestational age of 28 weeks or more, 2) prepartum adolescents and illiterate women who had obtained permission from their parent(s) or legal guardian(s) to participate in the study and 3) those who gave birth outside the hospital but were later admitted at HAM on the day of birth.The exclusion criteria included the following: 1) women with induced termination of pregnancy for medical reasons, 2) adolescent or illiterate mothers who had not obtained permission from their parents or legal guardians to participate in the study, 3) women without an ANC pregnancy card and 4) those missing information on the timing of first ANC contact.Of the 534 eligible participants, 89 women were excluded . A total of 445 pregnant women were included in this study.http://www.raosoft.com/samplesize.html). Using a minimum sample of 10% of the population, validated by the software, placed the sample right dimension between 355 (95%) and 579 (99%) confidence. This size was also supported by PASS software (https://www.ncss.com/software/pass/). It was possible to collect 445 women, which gave some comfort at this level. Eligible cases were selected randomly until the required sample size was achieved.The sample size was calculated using Raosoft software  executed and was responsible for all activities as follows: obtaining consent and enrollment of the mothers, data collection from ANC pregnancy cards, face-to-face interviews, and performing all data collection entry into the survey app.The outcome variable of interest was the utilization of ANC services. This utilization was classified as adequate or partial, based on the number of ANC contacts and first booking in the first trimester as well as the completion of hemoglobin, urine tests and obstetric ultrasound.Adequate ANC utilization was defined when all the following interventions were achieved: 1) attended ANC four or more times, 2) first booking during the first trimester, 3) had at least done one ultrasound, 4) had at least one urine test and 5) performed no less than one hemoglobin test.Partial ANC utilization category involved the remaining pregnant women, namely, those who attended antenatal clinic one or more times regardless of, whether they had or not, an obstetric ultrasound, an urine or any hemoglobin tests.In accordance with previous knowledge, the variables included in this study that could potentially be associated with ANC service utilization were age, residence, maternal education, husband/partner education, maternal occupational status, marriage, planned pregnancy, contraceptive utilization, and parity , 14, 19.Age was categorized as 14–19 years, 20–34 years and 35 years and above. Women’s education level has been included as a categorical variable ranging from no education, primary education, secondary and higher education, the same criteria for the baby’s father’s education. Household characteristics were characterized as having sanitation (toilet facilities or latrine) or not. Residence was grouped into urban and rural. Urban residence for women living at the capital city (Água Grande) and rural residence in all other districts. Employment status (working or not working) was defined as working for those who engaged in one economic activity. Parity was categorized as nulliparous, one to four parity and grand multipara (five or more). Marital status considered the two categories married/union vs single/never married. Women who were formally married or those who reported being in a union (cohabiting with a partner) were categorized as married/union.Data entry for this study into the app survey was first cleaned by creating a data entry field in Excel, and all categorical responses were checked for completeness and accuracy and to eliminate errors and inconsistent data. Data were further analyzed using the Statistical Package for the Social Sciences for Windows, version 25.0 .p value < 0.25). In all these analyses, logistic regression models for binary response were applied. Linearity in the logit assumption of age was verified with generalized additive regression models. The crude odds ratios (COR) and adjusted odds ratios (AOR) with corresponding 95% confidence intervals (95% CI) were then estimated. The level of significance α = 0.05 was considered. The goodness-of-fit of the multivariable model was checked with the Hosmer–Lemeshow test.Data analysis in this study was carried out in two stages. The first stage involved pooling data for descriptive statistics with categorical variables presented as frequencies and percentages, and quantitative variables presented as the mean and standard deviation. The second stage involved a univariable analysis to identify the candidate variables for the multivariable analysis  after the purpose of the research was explained orally by the main investigator. Additionally, if the mother was illiterate and unable to write, oral consent was given in the presence of at least one witness, usually the ward nurse´s complementarity to the mother´s fingerprint mark in the consent form. Participation in the survey was voluntary, as participants could decline to participate at any time during the study. The anonymity and safety of participants were ensured.Table A parity of one to four was observed in 271 (60.9%) pregnant women, with 136 (30.6%) of the participants being nulliparous. Unplanned pregnancy was reported in 252 (68.9%) cases, and previous contraceptive use occurred in 90 (24.5%) of the participants.Residency in an urban area was assessed for 202 (46.2%) participants, and 235 (53.8%) were living in rural districts, namely, 129 (30%) in Mé-Zochi, 48 (10.8%) in Cantagalo, 25 (5.6%) in Lobata, 17 (3.8%) in Lembá, 9 (2%) in Caué and 7 (1.6%) in Principe Island.Of the 445 participants, 378 (84.9%) had four or more ANC visits, 268 (60%) had a first booking during the first trimester, 214 (48%) pregnant women had at least one obstetric ultrasound, 280 (62.9%) had one urine test and 294 (66%) had a hemoglobin test.The results in Table Adequate ANC service utilization was identified for 213  pregnant women. Partial ANC service utilization was defined for the remaining 232  women.Of note, from the adequate ANC utilization group, a “complete” ANC utilization was found in 22 pregnant women who had all the following: attended ANC clinic eight or more times, first booking in the first trimester, had at least one ultrasound, had at least two urine tests, and performed no less than two hemoglobin tests.The results of the univariable analysis Table  showed aThe results indicate that in the unadjusted analysis the crude odds (COR) of adequate ANC utilization increased approximately twice for women who were within the age group of 20–34 years  and 35 years and above  compared to women in the 14–19 age group. The odds of having adequate ANC service utilization increased approximately twice for women from households with sanitation  and living in urban areas  and increased 2.68 times for women who planned the pregnancy .In Table p = 0.005) and those 35 years old or above , living in urban areas  and with a planned pregnancy  are more likely to have adequate ANC service utilization. A p value = 0.810 was obtained by the Hosmer–Lemeshow test indicating that the model was well calibrated.In the multivariable logistic regression, mothers’ age, residence, and pregnancy type of planning continued to have an association with adequate ANC service utilization. The results indicate that women in the 20–34-year age group  family planning methods as part of a system of traditions that is passed down from generation to generation through the teaching of certain beliefs , 10. We These risk factors were also analyzed in other studies conducted by the authors within the context of a broader research on the causes and risk factors contributing to neonatal mortality and adverse birth outcomes in Sao Tome & Principe –42.All the previous findings were obtained in the univariable study. However, in the multivariable analysis only some of the factors remained in the final model. Accordingly, determinants of an adequate ANC utilization in this study were maternal age greater than twenty years (sociodemographic characteristic), urban residence (community level characteristics), and a planned pregnancy (pregnancy related characteristic).This study included data with a total number of pregnant women similar to the one recently published for the country in 2019 Multiple Indicator Cluster Survey (MICS) from UNICEF . In contAnother main strength is that this study included essential routine screenings  in addition to the number of contacts per pregnant woman and an early booking during the first trimester. In contrast to most studies in SSA countries that only analyze ANC utilization in terms of the ANC number of contacts and/or timing , 13.This study was conducted in the capital city and may overestimate actual ANC visits as attitudes and practices may differ from rural areas in terms of access to health care, health worker motivation and training, and availability of health services. Factors such as women´s autonomy and the impact of media exposure were not assessed in this study, missing out some determinants associated to ANC utilization, such as sociocultural beliefs and convictions that sometimes dissuade women from accessing the antenatal care services .Further research is needed to determine the effects of traditional, sociocultural, religious, and other related practices at the community-level on the utilization of ANC services. Another important subject would be to determine the extent to which real family planning counseling takes place during ANC visits.Less than half of the pregnant women reached adequate ANC utilization, highlighting the challenge still ahead for ANC optimization in Sao Tome & Principe. The present study showed that the determinants of an adequate ANC utilization were maternal age greater than twenty years, urban residence, and a planned pregnancy. Understanding pregnant women´s determinants of ANC utilization in view of improving it is an excellent bridge to achieve its optimization. Measures such as raising awareness of the importance of ANC screenings completion, engaging the more vulnerable women in earlier utilization of family planning services, and promoting each women empowerment for a pregnancy plan are key strategies to improve neonatal health outcomes in the country.


Urinary incontinence (UI) is a common problem which is associated with impaired quality of life. Vitamin D plays a crucial role for pelvic floor muscle function. The aim of this study was to investigate the effect of vitamin D deficiency on UI in pregnant women in the third trimester of pregnancy. All pregnant women at > 28 weeks of gestation who were followed in the gynecology and obstetrics outpatient clinic were screened. The patients were assessed for UI during routine follow-up. The Incontinence Severity Index was used to determine the severity of UI. A total of 210 patients were included as the study group and 40 patients were included as the control group. Both groups were compared based on the International Incontinence Severity Index scores. Of the patients, 40% had a history of UI and 84% had vitamin D deficiency. Pregnant women with vitamin D deficiency had statistically significant UI, compared to pregnant women in the control group. The severity of UI was also significantly higher in the patients with vitamin D deficiency. Urinary incontinence is significantly associated with vitamin D deficiency in pregnant women. Pregnancy and vaginal delivery are major risk factors for UI. The mean prevalence of UI during pregnancy is 41%. Risk factors for the development of UI during pregnancy include weight gain increased uterine volume, increase intra-abdominal pressure, pressure on the pelvic floor and bladder, and enlargement of the genital hiatus with increasing parity.,5 The peripartum period is frequently the first time women experience UI. A study has shown that UI during pregnancy significantly increases the risk of developing UI within 12 years of delivery.Urinary incontinence (UI) is a serious health issue associated with anxiety, depression, and difficulties at work that contribute to social isolation.Vitamin D influences both male and female reproductive tissues via receptors. Via receptors are found in the hypothalamus, ovaries, testes, endometrium, and placenta. Therefore, vitamin D plays an important role in human reproductive physiology.,9Vitamin D is important for couples fertility, and Vitamin D deficiency could be a cause of infertility and affect the success of assisted reproductive techniques. Vitamin D deficiency and insufficiency may have a significant negative effect on pregnancy outcomes. Several gestational complications, such as gestational diabetes, gestational hypertension, premature rupture of membranes, and premature delivery may occur due to vitamin D deficiency.Vitamin D affects decidualization, implantation, and hormonal and immune responses in the placenta.,13 In addition, vitamin D deficiency during pregnancy has been linked to postpartum pelvic floor muscle dysfunction. To the best of our knowledge, only 1 study investigating the association between vitamin D deficiency and UI during pregnancy. Stafne et al, in their study including 851 healthy pregnant women between 18 and 22 weeks of pregnancy, reported that UI in pregnant women with vitamin D deficiency was significantly more common with a higher prevalence.Vitamin D also plays a crucial role in pelvic floor muscle functions. Several studies have demonstrated that vitamin D deficiency is associated with UI in nonpregnant women.In this study, we aimed to investigate the prevalence of UI and examine the relationship between vitamin D deficiency and UI in full-term pregnant women.This single-center, cross-sectional study was conducted at the Department of Gynecology and Obstetrics of a tertiary care center between January 2021 and November 2021. The study included 250 healthy pregnant women over the age of 18 years with a singleton pregnancy over 28 weeks who were admitted to our clinic  (Appendix 1: ISI index). The ISI includes 2 subitems: “How frequently do you urinate?” and “How much urine do you leak per day?”. The ISI score was calculated by multiplying the number of questions for each item. The score was determined by grouping 1 and 2 as mild, 3 and 6 as moderate, 8 and 9 as severe, and 12 as extremely severe (Appendix 1).Demographic data, obstetric histories, smoking, alcohol use, drug use, and chronic medical conditions were recorded. During antenatal follow-up, pregnant women who were at ≥ 28 weeks of pregnancy were questioned about their UI history. Subjective incontinence was deemed positive if participants experienced UI at least once during pregnancy and negative if they did not. The International Incontinence Severity Index (ISI), developed by Sandvik et alDuring the initial evaluation, 5 cc of venous blood was drawn from the pregnant women, centrifuged, and stored at −80°C until analysis. By requesting a thorough urinalysis and, if necessary, urine culture, an acute urinary infection was ruled out. Serum vitamin D levels were measured after the completion of 250 cases. Serum 25 (OH) D3 concentrations were measured using enzyme-linked immunosorbent assay . Serum 25 (OH) D3 concentrations < 20 ng/mL were classified as deficient . The study group consisted of 210 women with insufficient vitamin D levels, whereas the control group consisted of 40 women with adequate vitamin D levels. Both groups were compared based on ISI scores.t test was used to compare the groups, whereas the Mann–Whitney U test was used for non-normally distributed variables. Fisher chi-squared test was used to compare categorical variables. If the Fisher exact test result was significant, the 2-ratio Bonferroni-corrected z test was performed to analyze the differences between the groups. Statistical significance was set at P value < .05.Statistical analysis was performed using the SPSS version 26.0 software . Descriptive data are presented as mean ± standard deviation or median (Q1–Q3) for continuous variables and as numbers and frequencies for categorical variables. The normality of the data distribution for numerical variables was assessed using the Shapiro–Wilk test and Q-Q charts. Levene test was used to assess homogeneity of variance. For normally distributed variables, a 2-sample independent 2 and 27.82 ± 3.93 kg/m2, respectively. The mean gestational week was 33.86 ± 4.11 weeks, respectively. Both groups were similar in terms of age, body mass index, and gestational age.Baseline demographic characteristics of the participants are presented in Table A total of 40% (100) of all the pregnant women who participated in the study had UI at any time during pregnancy. On comparing the 2 groups based on subjective incontinence reporting, 42.4% (n = 89) of the pregnant women in the study group and 27.5% (n = 11) of the pregnant women in the control group experienced UI at least once during pregnancy (Table P = .026). The ISI scores of the pregnant women in the study group were also significantly higher than those of the control group (Table A total of 42.4% of pregnant women with vitamin D deficiency had UI compared to 27,5% of pregnant women in the control group (In the present study, we investigated the effect of vitamin D deficiency on UI in women in the third trimester of pregnancy. Our study results showed that 84% of the women had vitamin D deficiency, while 40% had a history of urinary incontinence. Pregnant women with low vitamin D levels had significantly more UI issues than those with normal vitamin D levels. In a recent study conducted in Turkey, the prevalence of vitamin D deficiency in individuals aged 2152 years was estimated at 40% to 96.6%.Vitamin D deficiency during pregnancy is a global health issue.,21 It has been shown in the literature that the prevalence of urinary incontinence is lower in patients with high vitamin D levels.,13,22,23 During the postpartum period, a significant decrease in pelvic floor muscle strength has been observed in patients with vitamin D deficiency. In addition, the incidence of UI was higher among women with vitamin D deficiency, although the difference was not statistical significance. A recent study showed that vitamin D replacement reduced the severity of SUI in women with vitamin D deficiency.The pelvic floor muscles and bladder contain vitamin D receptors. Therefore, vitamin D deficiency may affect pelvic floor function and urinary continence. In this study, Stafne e al. found that 27% of healthy, nulliparous, mid-trimester pregnant women living in Northern Europe experienced UI, particularly stress incontinence, and the prevalence of UI was higher in pregnant women with vitamin D deficiency.To the best of our knowledge, only 1 study has investigated the relationship between vitamin D deficiency and UI during pregnancy in the literature.In the present study, consistent with the literature, but slightly higher than that reported by Stafne et al, we determined that the prevalence of UI in pregnant women living in our region was 40%. To evaluate the relationship between vitamin D deficiency and UI during pregnancy, we assessed the presence and severity of UI in pregnant women with vitamin D deficiency. We aimed to determine the serum vitamin D levels of women in the third trimester of pregnancy to reduce the effect of factors such as nutrition, dressing style, and supplementation status on vitamin D levels in pregnant women. We found that both the presence and severity of UI were significantly higher in pregnant women with vitamin D deficiency than in the control group. Considering the high prevalence of pelvic floor disorders and incontinence in the female population with vitamin D deficiency, we believe that it is reasonable to have similar results in pregnant women with vitamin D deficiency.Nonetheless, this study had some limitations. The main limitations of this study its cross-sectional design and relatively small sample size. On the other hand, the main strength of our study is that it investigated both the current UI status and UI experience during pregnancy in pregnant women, and it is one of the first studies to examine the association between vitamin D deficiency and UI during pregnancy.In conclusion, vitamin D deficiency is significantly associated with the development of UI in pregnant women. Although these results are consistent with those in the existing literature, further large-scale prospective studies are required to investigate the association between vitamin D deficiency and UI in pregnant women.Conceptualization: Sezer Gul.Data curation: Sezer Gul, Huseyin Aydogmus, Caglasu Su, Serpil Aydogmus.Formal analysis: Sezer Gul, Huseyin Aydogmus.Investigation: Sezer Gul, Huseyin Aydogmus.Methodology: Sezer Gul, Huseyin Aydogmus.Resources: Sezer Gul.Supervision: Mustafa Sengul.Validation: Sezer Gul.Writing – original draft: Sezer Gul, Huseyin Aydogmus, Mustafa Sengul.Writing – review & editing: Huseyin Aydogmus, Serpil Aydogmus, Mustafa Sengul.


Previous studies have shown that mice fed a diet containing 1% mantle tissue exhibited decreased food consumption and led to death. Toxic substances present in the mantle tissue have been isolated and identified. In the present study, we explored the characteristics and stability of mantle tissue toxicity. The treatment of mantle tissue with 1 mM hydrochloric acid, 1 mM sodium hydroxide, 1 mM dithiothreitol, and 1 mM hydrogen peroxide followed by heating did not significantly reduce the toxicity of mantle tissue in mice. These results suggest that mantle toxins are stable in tissues, particularly when exposed to acidic conditions and digestive enzymes. We examined whether mantle tissue exhibited acute toxicity. Mice fed a diet containing 20% mantle tissue did not show a distinct increase in toxicity compared with mice fed a diet containing 1% mantle tissue, demonstrating that feeding mantle tissue does not lead to acute toxicity. Finally, mantle tissue toxicity in the small intestine was examined. Chronic feeding of mantle tissue to mice changed the color of the small intestine. Real-time polymerase chain reaction analysis revealed that mantle tissue feeding caused changes in inflammation and endoplasmic reticulum stress markers in the small intestine. These results suggest that mantle tissue feeding causes toxicity after initial damage to the small intestinal tissue. Bivalve mollusks, such as scallops, mussels, and clams, accumulate toxins in their digestive glands by consuming toxic microalgae ,2. ParalIn Japan, scallop toxins are likely to occur during summer and fall, and paralytic and diarrhetic shellfish toxins are detected in different regions annually. In Japan, to minimize the risk of shellfish poisoning, the Ministry of Agriculture, Forestry, and Fisheries, as well as local authorities, conduct seawater monitoring and shellfish inspections. If shellfish toxins are detected, fishing and sales may be restricted, ensuring the protection of public health and enabling the safe consumption of shellfish.Scallop is a significant marine product in Hokkaido, Japan, resulting in the generation of approximately 300,000 tons per year of scallop shells as industrial waste. For the effective utilization of the scallop shells, bioactivities of organic components in scallop shells have been extensively investigated ,7,8,9,10Furthermore, a novel scallop toxin comprising a complex of the N-terminal region of a gelsolin-like protein with a molecular weight of 18 kDa and actin fragments with a molecular weight of 25 kDa was isolated. Gelsolin-like protein is expressed in the mantle tissue, ovary, and gill tissue, but not in the adductor muscle, testis, or digestive glands . ToxicitRicinus communis L (CB), which are isolated from plants, exhibit acute toxicity ) at pH 7 at 37 °C overnight. After treatment, each sample was freeze-dried and subjected to a toxicity evaluation. Samples containing only proteases without mantle extract were used as controls.To evaluate the stability of toxicity of the mantle extract against digestive enzymes, the mantle extract was treated with pepsin  mice were purchased from CLEA . The mice were housed individually in a room maintained at 22 °C. Three or four mice were housed in together in cages and maintained at 22 °C with free access to water and food. The mice were acclimatized for at least 7 d and used for each experiment. Mice were fed either a normal AIN-93G-based diet (control diet) or an AIN-93G-based diet containing 1% mantle tissue or 1–20% mantle extract at 4 g/d for 4–12 weeks . The prog for 15 min at 4 °C. The supernatant was used as the small intestine extract.The small intestine and cecum were isolated 3 weeks after the mice started consuming a diet containing mantle tissue or a control diet. Small intestine (1 mg) was homogenized in 20 mL of deionized water and centrifuged at 12,000× Animal experiments were conducted in accordance with the Guidelines for Experimental Animal Care issued by the Office of the Prime Minister of Japan and the Muroran Institute of Technology. All experiments were approved by the Committee on Ethics, Care, and Use of Animal Experiments of the Muroran Institute of Technology (Permit Number: H29-KS04). The health status of all the mice was monitored by assessing their daily food and water intake and observing their appearance during the experimental period. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously . BrieflyTotal RNA was extracted from small intestinal tissues using the RNAiso Plus Kit . First-strand cDNA was synthesized using 10 μg of RNA and an oligo (dT) primer. Real-time polymerase chain reaction (PCR) was performed using iTaq Universal SYBR Green Supermix  , with 25g for 1 min at 25 °C. Thiobarbituric acid (TBA) solution (0.67%) was incubated with the supernatant at 100 °C for 1 min, and absorbance at 540 nm was measured. Malondialdehyde levels, a marker of oxidative stress, were evaluated using thiobarbituric acid as previously described ,23. Brieg for 1 min at 25 °C, 5 μL of 20 mM 2-ethylbutyric acid  solution and 5 μL of 35% HCl solution were added to the supernatant. Diethyl ether (200 μL) was added to the mixed solution and centrifuged at 12,000× g for 1 min at 25 °C. The upper layer was used to measure the SCFA contents using GC with a GC-4000 GC system  equipped with a flame ionization detector (FID) and a capillary column . The temperature program was set as follows: 160 °C for 2 min, followed by a linear gradient from 160 °C to 300 °C at a rate of 20 °C/min. The total analysis time was 10  min. The concentration of SCFAs in the cecal contents was determined using gas chromatography (GC) . BrieflyThe toxicity of scallops under various conditions was assessed based on the decrease in food intake. Data on food intake from three mice per group are expressed as the mean ± standard deviation (SD). Food intake of the dead mice was recorded as zero for the calculations.p < 0.05.Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the Tukey–Kramer multiple comparison test using BellCurve for Excel ver. 2.15 . Statistical significance was set at t-test. Statistical significance was set at p < 0.05. Each experiment was performed at least twice to ensure reproducibility.Data from real-time PCR, SCFA contents, and malondialdehyde (MDA) contents in the small intestine were obtained using twelve mice and expressed as the mean ± SD. Statistical analysis was performed using Student’s two-tailed 2O2 or 1 mM dithiothreitol exhibited a decrease in food intake starting in the third week and died in the fourth or fifth weeks, similar to mice fed the mantle diet. After treating the mantle tissue at 100 °C for 20 min, food intake did not change significantly between mice fed the diet containing heat-treated mantle and those fed the mantle diet at the first, second, fourth, and fifth weeks.Toxicity in mice was evaluated by comparing the decrease in food intake, reflecting the toxicity of the mantle tissue, as described previously . The staThese results suggest that the toxic substances were stable within the mantle tissue.Furthermore, we examined the stability of mantle extract toxicity a. In mice fed a diet containing NaOH-treated mantle extract, a decrease in food intake was observed in the fourth and fifth weeks, which recovered after the sixth week; the mice did not die until the seventh week. After heating, the toxicity of the mantle extract was reduced, whereas treatment with 1 mM HCl had no effect on mantle extract toxicity. Food intake did not change significantly during the first to fourth weeks between mice fed the diet containing HCl-treated mantle extract and those on the mantle diet. This result was consistent with our previous study , which rFinally, the toxicities of three smoked mantles produced by various companies were evaluated. The mantle diet showed a decrease in food intake starting in the second week, and mice died in the fourth week. One (Product A) of the processed mantle food exhibited a decrease in food intake and mice died after 8 weeks, although the toxicity was weaker than that of the mantle tissue. A significant difference in food intake from the fourth week was observed between mice fed a diet containing Product A and those fed the mantle diet, whereas products B and C showed no toxicity d.To examine whether toxic substances in the mantle tissue exhibited acute toxicity, mice were fed diets containing different amounts of mantle extract  a. Mice fTo investigate whether a toxic substance accumulated in tissues, we examined the toxicity when mantle tissue was administered to mice every other day, every 2 days, and once weekly b. When aNext, we investigated whether toxicity was observed after the mice were fed a diet containing 1% mantle tissue for two weeks, followed by switching to a control diet c. Even aFinally, we determined whether the toxicity of scallop mantle tissue varied depending on the location and season of collection a,b. All A previous study has demonstrated that a diet containing scallop mantle tissue leads to liver and kidney injury. In the present study, we focused on mantle tissue toxicity in the small intestine. Mice fed a diet containing 1% mantle tissue for 3 weeks showed a change in the color of the small intestine a. LevelsIn the small intestines of mice fed mantle tissue, there were significant increases in the levels of peroxidized lipids a, and thIn the present study, we compared the mantle tissue toxicity in mice by assessing food intake as an indicator of toxicity. A decrease in food consumption was frequently noticed within 2–3 weeks, resulting in death of the mice between the third and fifth weeks. However, the intensity of toxicity and food intake varied considerably between the experiments. Although the exact reasons remain unclear, this variability may be significantly influenced by the conditions of the purchased mice and the state of the scallops , even whOur previous study showed that the toxic substance in mantle tissue is a protein complex . AlthougThe ricin toxin exhibits toxicity even after oral administration . SimilarBacteroides fragilis induce inflammation in the small intestine and disrupt barrier function [Several studies have reported that resistant proteins are resistant to digestive enzymes and behave as dietary fibers, similarly to cellulose, pectin, gum, and lignin ,32. Addifunction . SimilarClostridium toxin A, which causes inflammation in the human small intestine, affects the actin cytoskeleton of small intestinal epithelial cells, resulting in barrier dysfunction [In a previous study, a complex of gelsolin-like proteins and actin was isolated from scallops as a novel toxin that acts on the actin cytoskeleton in cells, causing changes in cell morphology and exhibiting toxicity . Clostrifunction ,40. ToxiWhen the mice were fed a diet containing 20% or 1% mantle extract, no distinct decrease in food intake was observed. This result indicates that even the ingestion of large amounts of mantle tissue does not cause acute toxicity. The amount of toxins absorbed into the small intestinal tissues may be limited. Furthermore, when mantle tissue was ingested every other day, toxicity was observed. However, when ingested every 2 d, no toxicity was observed. It may be necessary for a certain quantity of toxic substances to be present within the small intestine and continuously act on intestinal epithelial cells to cause toxicity. The toxicity of mantle tissues occurs with continuous ingestion; therefore, toxin in the mantle tissue does not appear to accumulate gradually in the intestines, liver, and kidneys. Lingula anatina and Mercenaria mercenaria have gelsolin-like proteins with high homology values of 86% and 82%, respectively, to scallop gelsolin-like proteins, as shown in the BLAST search. These shellfishes are also consumed in Japan. Therefore, further investigations into various processed products and shellfish are required.The toxicity of the scallop mantle tissue was not dependent on the location or season of collection. The expression levels of a previously identified toxic substance, gelsolin-like protein, may not vary significantly with location or season. In this study, we focused on scallop mantle tissue. Our previous study reported that the scallop ovarian tissue, which is often consumed in Japan, is toxic. This toxin may also be present in other shellfish species. Whether the ingestion of mantle tissue causes toxicity in humans has not been reported, likely because feeding mantle tissue does not cause acute toxicity, and the continuous ingestion of mantle tissue is required to induce toxicity. However, the possibility that the chronic ingestion of mantle tissue affects human health, including intestinal inflammation, and kidney and liver injury, cannot be excluded. Currently, a search for various substances that can alleviate this toxicity and elucidate the pathways necessary for the expression of toxicity in mantle tissues is underway.Toxicity of the mantle tissue persists even after acid, base, and heat treatments, indicating the marked stability of the toxic substance in the mantle tissue. Toxicity was observed when mantle tissue was consumed continuously, and the ingestion of a large amount of mantle tissue did not result in acute toxicity. Additionally, changing to a control diet after feeding the mice mantle tissue led to a decrease in food intake and death. The present results suggest that the continuous ingestion of mantle tissue triggers inflammation, ER stress, and oxidative stress in the small intestine, leading to the incorporation of toxic substances into the liver, kidneys, and other parts of the body. In the future, epidemiological research should be performed to determine whether the intake of scallop mantle and ovaries, which contain new potential toxins, influences human health, specifically its correlation with the incidence of intestinal inflammation, liver disease, and kidney disease.


Chenopodium album, Echinochloa crus-galli and Portulaca oleracea) and three crops  was investigated.Weeds represent a great constraint for agricultural production due to their remarkable adaptability and their ability to compete with crops. Climate change exacerbates the abiotic stresses that plants encounter. Therefore, studying plant responses to adverse conditions is extremely important. Here, the response to saline stress at different temperatures of three weed species (C. album was not significantly affected by salinity, but it decreased in E. crus-galli and P. oleracea with increasing salinity. The mean germination time for all species increased with salinity, especially at lower temperatures. This effect was most pronounced for soybean and E. crus-galli. C. album exhibited significant reduction in stem growth with high salinity and high temperatures, while in E. crus-galli stem growth was less reduced under similar conditions.The germination percentage of soybean notably decreased as salinity and low temperatures increased. In contrast, maize and rice consistently maintained a high germination percentage, particularly when subjected to low salinity levels. Regarding weed species, the germination percentage of This study showed that successful germination under saline stress did not ensure successful early development and emphasizes the species-specific nature of the temperature-salinity interaction, perhaps influenced by intraspecific variability. Increasing salinity levels negatively impacted germination and seedling growth in most species, yet higher temperatures partially alleviated these effects.The online version contains supplementary material available at 10.1186/s12870-023-04465-8. Salt-affected soils  are defined as the soils containing high concentrations of soluble salts, which adversely affect the growth and productivity of most crops , 2. The 2 influx due to stomatal closure E. crus-gddition,  found suC. album) showed significantly reduced root and stem lengths with increasing salinity. This discrepancy can be explained by the fact that many plant species exhibit different responses to salt stress depending on the growth stage, among which are the activation of salt-stress responsive genes, the induction of Na+ and Cl− efflux root transporters, the accumulation of osmolytes and Na+ vacuolar sequestration [The effects of salinity and temperature at the early growth stage were on the whole consistent with the effects on seed germination, with a few exceptions. Those species that appeared to be salt tolerant at the germination stage  or in shoot growth and root elongation . This finding suggests that these weed species could be important competitors with crops grown on saline or salt-affected soils, especially at higher temperatures, intensifying their negative effects on crop development. On the other hand, two of the crop species tested, maize and rice, also showed tolerance to high salinity levels at the germination and growth stages. In view of this, one of the ways to suppress or reduce crop-weed competition in saline soils might be to grow salinity-tolerant crop cultivars, given that crops usually tend to germinate at a homogeneous rate, grow fast, and create large canopies that shade the weeds. This can be seen in the case of maize, the germination and growth of which seem to be stimulated by low salinity concentrations.The most detrimental combination was high salinity and low temperature. The weed species tested had a high degree of salt tolerance either as a reduction in the low germination percentage  and osmoprotectants to crops could be valuable approaches to increase the crop tolerance to salt stress, thereby making them more competitive against weeds.More studies should investigate (i) the germination and seedling response of other weed species that could infest crops grown on saline soils, and (ii) the competition between crops and different weed species in the further stages of development. In this regard, soil experiments in controlled saline conditions could confirm our hypothesis of increased weed competitiveness in saline environments. In addition, studies concerning the influence of salinity on soil seedbank might give us further insights into possible development of future weed infestation.Chenopodium album, Portulaca oleracea, and Echinochloa crus-galli, common in the fields of soybean, rice and maize of Northern Italy, were collected from September to November 2020 at the Experimental Farm of the University of Padua in Legnaro . They were hand-harvested on warm dry days by shaking into paper bags to ensure that only mature seeds were collected. The seeds were then hand cleaned and stored in paper bags at room temperature until the start of trials the following spring. For the crops, three major species were chosen: maize (Zea mays) variety DKC5530, soybean (Glycine max) variety P21T45, and rice (Oryza sativa) variety Vialone Nano.Mature seeds of three summer weed species, The saline solutions were prepared by adding pure sodium chloride (NaCl) to distilled water until the desired salinity level was reached, which was measured with an XS Instruments COND 80 electrical conductivity meter  at a sensitivity of 1 µS. Four different saline solutions were prepared: 4, 8, 12 and 16 dS/m, in order to reach these salinity levels 3.20 g/l; 5,50 g/l; 8,50 g/l and 12, 80 g/l of NaCl were added to distilled water. The control consisted of pure distilled water (0 dS/m).E. crus-galli showed some degree of dormancy (data not shown). E. crus-galli is known for having dormancy that can be overcome by seed scarification with sulfuric acid [E. crus-galli were immersed in 98% sulfuric acid for 20 min and then thoroughly rinsed. In order to prevent imbibition of the seeds with water, those meant for the trials with saline solutions were rinsed with those solutions after the acid scarification process.Germination tests for each species, treated with NaCl and untreated, were conducted in three climate chambers with a 12 h light/12 h dark photoperiod, and three different constant temperatures of 12 °C, 15 and 18 °C, simulating early spring conditions of the soil. Four biological replicates (1 replicate = 1 plate) were used for each species at each salinity/temperature combination. Therefore, 60 replicates were prepared for each species . Each replicate consisted of 100 seeds in the case of the weeds, 50 in the case of the crops. The seeds were placed in 9 cm-diameter Petri dishes (14 cm-diameter for maize and soybean because of the larger seed size) lined with filter paper and moistened till filter paper was fully imbibed with saline solution (or distilled water for the controls). After placing the seeds on filter paper, the Petri dishes were sealed with parafilm and set inside the climate chambers according to a randomized design. Their position was exchanged every other day. Germination was monitored every 2–3 days, the seeds were considered germinated when a radicle of 1 mm or longer was developed, and was considered completed if all the seeds germinated or if 10 days elapsed without germination, as proposed by . Upon coric acid . TherefoTo assess the effects of salinity and temperature on early seedling development, growth tests were conducted by sowing seeds in half-strength MS agar medium  without In line with the size of the containers and the size of the seeds, 20 crop seeds and 50 weed seeds were sown per container. Salinity and temperature levels were the same as for the germination test, and four replicates were used for each species at each salinity/temperature combination. To prevent contamination of the agar medium, sowing took place inside a sterile environment under a laminar flow hood, and prior to sowing all seeds were sterilized for 30 s with 75% ethanol, followed by a 15-minute treatment with 15% (v/v) sodium hypochlorite (NaClO). The seeds were then washed in distilled water for 5 × 5 min. Once the sowing was completed, the containers were placed inside the climate chambers. In accordance with their different growing speeds, the growth of the crop species was measured after two weeks, the weed species after five weeks. After the established growth period, the plants were carefully removed from the containers, and their stem and root elongation were measured with a digital caliper .For the germination tests, mean germination time (MGT) was calculated using the formula proposed by :MGT = Σ(nD) / Σn.where D is the number of days since the start of the test, and n is the number of newly germinated seeds at day D, in accordance also with . The effBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7