This study aims to evaluate the value of the clinical application of ultrasound-guided percutaneous thermal ablation in focal nodular hyperplasia (FNH) by comparing its safety, effectiveness, and patient experience to surgery in the treatment of hepatic FNH ≤5 cm.This retrospective study enrolled 82 patients with hepatic FNH having a maximum diameter of ≤5 cm, confirmed by postoperative pathologic diagnosis or needle biopsy, who underwent thermal ablation or surgery between January 2019 and September 2021. Postoperative efficacy, surgical trauma , postoperative complications , patient experience , and economic indices  were compared between both groups.p > 0.05). No recurrent or new lesions were observed during the 6-month follow-up in both groups. However, significant differences were observed in operation time, intraoperative bleeding volume, and lost volume of normal liver tissue (p < 0.05), with significantly less trauma in the thermal ablation group. No statistically significant differences in ALT, AST, and Hb existed between both groups (p > 0.05); however, albumin was higher in the ablation group compared to the surgery group , and WBC were lower in the ablation group . The incidence of postoperative complications in the ablation group was significantly lower than that in the surgery group (p < 0.05). Patient experiences were significantly better than in the surgical group (p < 0.05), with economic indicators being significantly less in the ablation group (p < 0.05).No significant difference existed in postoperative efficacy between both groups (Ultrasound-guided percutaneous thermal ablation can treat hepatic FNH ≤5 cm with similar clinical efficacy as surgery and is an economical, safe, and minimally invasive treatment method worthy of recommendation. Hepatic focal nodular hyperplasia (FNH), a proliferative lesion in which the liver parenchyma proliferates and is separated into nodules by stellate fibrous scars (WHO criteria) , has an Since most FNH patients have normal laboratory values with no special clinical manifestations, diagnosis is mainly based on imaging examination. In recent years, with improvement in imaging techniques and deepened understanding of FNH by clinicians, its diagnostic level has significantly improved. ACG clinical practice guidelines on the management of focal liver lesions  propose n = 53) or surgery (n = 50) in Fujian Provincial Hospital and the Third Affiliated Hospital of Guangzhou Sun Yat-Sen University between January 2019 and September 2021 were reviewed. “Lost to follow-up” was defined as failure to obtain complete follow-up data. Patients were included based on the inclusion and exclusion criteria, and patients lost to follow-up were excluded. A total of 82 patients  met the inclusion criteria, of which 39  with 49 lesions  underwent thermal ablation  and 43  with 50 lesions  underwent surgery (surgery group).This study was approved by the ethical and scientific review board of Fujian Provincial Hospital. Medical records of 103 patients with hepatic FNH with a maximum diameter of ≤5 cm confirmed by postoperative pathologic diagnosis or needle biopsy, who underwent thermal ablation (The inclusion criteria were as follows: (1) patients with FNH confirmed by postoperative pathology or biopsy; (2) FNH lesion diameter ≤5 cm; (3) normal preoperative coagulation; and (4) clear preoperative lesion and number imaging.The exclusion criteria were as follows: (1) patients unwilling to undergo surgery or thermal ablation; (2) FNH lesion diameter >5 cm; (3) patients with other important organ diseases requiring combined operation; and (4) incomplete follow-up data.Thermal ablation was performed using a Viva RFA system  with a disposable monopolar RF ablation needle electrode  or a Microwave Ablation Therapy Instrument  with a disposable microwave ablation needle . Ultrasonography and contrast-enhanced ultrasonography were performed using the Philips IU22 with C5–2 low-frequency convex array probe to determine the specific location, number, and size of the lesion; also, an RFA or microwave ablation pathway was designed. The patient was placed in a supine position with the upper abdomen fully exposed. Sufentanil at 0.01–0.02 µg/kg and midazolam at 0.02–0.03 mg/kg were intravenously administered for sedation and analgesia, then the skin was disinfected and covered with a sterilized towel. In total, 2% lidocaine was used to locally infiltrate the skin at the anesthesia puncture site of the anterior capsule of the liver. The RFA or microwave ablation needle was inserted into the intrahepatic lesions. The ablative power was set at 100–120 W; the average temperature was controlled at 90°C and starting from the posterior and lower part of the lesion, gradual forward advancement was done needle-by-needle. Tumors with a diameter of <2 cm were ablated using point-one needle ablation, and tumors with a diameter of >2 cm were ablated by multipoint and multineedle ablation. For those clearly diagnosed on imaging, the inactivation range should cover the tumor completely; meanwhile, for those with no obvious diagnosis, the inactivation range should be 0.5–1 cm larger than the outer edge of the tumor  was determined according to the preoperative imaging examination and intraoperative exploration. An intraoperative ultrasound-guided ultrasonic scalpel combined with an electric hook was used to resect the liver tumor tissue while maintaining hemostasis. After complete wound hemostasis, an abdominal drainage tube was placed. The laparoscopic surgical resection of the left hemi-liver is shown in Follow-up observation indices were as follows: (1) basic information: patient’s gender and age, tumor diameter, and reasons for the choice of treatment; (2) patient trauma: preoperative and postoperative ALT, AST, albumin, Hb, WBC, operation time, intraoperative blood loss, intraoperative blood transfusion rate, and normal liver tissue loss volume; (3) postoperative complications: postoperative infection, pleural effusion, and severe liver dysfunction; (4) patient experience: postoperative fasting time, indwelling abdominal drainage tubes, scar size, degree and time of postoperative pain, length of hospital stay, and total hospitalization expenses; and (5) efficacy evaluation: lesion disappearance or inactivation and MR- or CT-enhanced follow-up.The curative effect was evaluated as follows: (1) cure: all target lesions disappeared  or were completely inactivated , and there were no new lesions within at least 6 months; (2) improved: the target lesions were partially residual or partially active ; and (3) invalid: volume of unresected or residual active lesions were >50%. During the 6-month follow-up period, MR-enhanced assessment was used, and follow-up contents included the presence or absence of new lesions and the residual activity of target lesions.3 and <10 cm3.All patients used CT three-dimensional visualization software (IQQA-LIVER) to perform retrospective three-dimensional reconstruction and calculate the volume of the preoperative liver tumor and nonenhanced area after ablation in the ablation group and the preoperative and postoperative liver and preoperative liver tumor in the surgical group. Normal liver tissue loss volume in the surgery group = preoperative liver volume − postoperative liver volume − preoperative liver tumor volume. Normal liver tissue loss volume in the ablation group = volume of the nonenhanced area after ablation − preoperative liver tumor volume. According to the calculation results, it was divided into two groups of ≥10cmt-test was used for the statistical comparison of measurements. Fisher’s Chi-square test or χ test was used for comparison between count data and categorical variables. p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS v19.0. The data were reported as mean ± standard deviation. The Student’s p > 0.05). The baseline characteristics of each group are summarized in In total, 82 cases were enrolled in this study, including 43 cases (18 men and 25 women) in the surgical group with 50 lesions and a mean age of 32.05 ± 8.8 years old; 39 cases  formed the thermal ablation group with 49 lesions and mean age of 35 ± 11.5 years. There were no significant differences in gender, age, preoperative maximum tumor diameter, and preoperative laboratory indicators  between both groups . The nature of the lesions could not be identified by clinical assessment and imaging or the possibility of malignant lesions could not be excluded, accounting for 29.3% (24/82). Among them, 10 had hepatitis cirrhosis and the first diagnosis was liver cancer. Four out of 82 patients had lesion enlargement during follow-up observation (4.9%) and seven out of 82 patients had clinical symptoms (8.53%).p > 0.05)  and ≥10 cm3 . In the thermal ablation group, the average operation time was 44.87± 13.3 min; intraoperative blood loss was as follows: bleeding volume <100 ml  and 100–500 ml . No intraoperative bleeding >500 ml or blood transfusion occurred; normal liver tissue loss was as follows: volume <10 cm3  and ≥10 cm3 . There were three patients in the thermal ablation group with intraoperative needle trace hemorrhage, of whom two had liver needle trace hemorrhage and bleeding volume was approximately 100 ml. After needle trace ablation, the bleeding stopped. Another case of hemorrhage occurred in the chest wall. Contrast-enhanced ultrasound (CEUS) can clearly detect chest wall hemorrhage and flow to the right thoracic cavity. The amount of blood loss was approximately 400 ml. Thrombin was injected into the bleeding point to successfully stop the bleeding. No continuous bleeding was observed for 2 days. In the surgery group, 16 patients (37.2%) had intraoperative blood loss >100 ml—two patients had bleeding >500 ml that required intraoperative blood transfusion and seriously increased body trauma. The operation time, blood loss, and normal liver tissue loss in the thermal ablation group were lower than those in the surgery group, and the differences were statistically significant (p <0.05) , 100–500 ml , and >500 ml . Two patients received an intraoperative blood transfusion at a rate of approximately 4.65%; normal liver tissue loss was as follows: volume <10 cmp >0.05). However, postoperative albumin was higher in the thermal ablation group compared to the surgical operation group, while postoperative WBC was lower in the ablation group compared to the surgical operation group; the difference between both groups was statistically significant (p <0.05). This comparison is shown in The postoperative laboratory indicators, ALT, AST, albumin, Hb, and WBC, of both groups, were statistically analyzed. There was no significant difference in postoperative ALT, AST, and Hb between the two groups , and the incidence of complications in the ablation group was much lower than that in the surgery group. In the surgical operation group, there were two cases of infection, one case of severe intracranial and cheek infection, and one case of incision infection, all of which improved after treatment; there were eight cases of postoperative pleural effusion, including two cases of pulmonary infection, six cases of postoperative liver dysfunction (two cases were severe liver dysfunction ALT >500 U/L). In the postoperative ablation group, two cases had pleural effusion and two had severe liver dysfunction (ALT >500 U/L)  and 10.2% (4/39), respectively. The difference was statistically significant (p < 0.05) .The postoperative hospital stay and total hospital expenses of patients in the ablation group were significantly lower than those in the surgery group , with significant differences between both groups  with suspected FNH but unclear diagnosis whose diagnoses were confirmed by surgery or ablation, 10 had hepatitis cirrhosis and the first diagnosis was liver cancer. Seven patients  were actively treated for clinical symptoms such as upper abdominal pain. Due to high psychological pressure, although FNH has been clearly diagnosed on imaging, 47 patients  who failed to accept this intrahepatic space-occupying lesion only needed treatment observation. They refused diagnosis of an intrahepatic spaceoccupying lesion and strongly desired treatment and 4.9% patients (4/82) with lesion enlargement at follow-up observation underwent thermal ablation or surgical treatment. Patients who cannot accept intrahepatic mass and are strong-willed regarding treatment are the main reasons for FNH treatment. Nonactive treatment of such patients will cause serious psychological problems such as anxiety, which do not favor physical and mental health. Providing a more safe and more effective treatment with less trauma for patients who need active treatment for FNH is worth considering.In recent years, with development in ultrasound diagnosis and treatment technology, ultrasound-guided thermal ablation has been widely used in the minimally invasive treatment of solid liver tumors and has achieved good therapeutic effects, which have been unanimously recognized by clinicians and patients. The Guidelines for Diagnosis and Treatment of Primary Liver Cancer (2022 Edition)  clearly p < 0.05). Both thermal ablation and surgical treatment of FNH can cause transient changes in the body’s laboratory parameters; however, the degree of injury is usually mild and usually returns and normalizes within a week. There was no significant difference in postoperative ALT, AST, and Hb between both groups; however, the albumin content in the postoperative surgical group was significantly lower than that in the thermal ablation group (p< 0.05). It showed that compared to thermal ablation, surgery results in greater nutrient loss.In this study, the operation time, anesthesia method, intraoperative blood loss, postoperative laboratory examination, and normal liver tissue loss were compared and analyzed to evaluate trauma from thermal ablation and surgery in the treatment of FNH. The trauma analysis of the two FNH treatment methods showed that thermal ablation was significantly less traumatic than surgery. In this study, the total operation time with thermal ablation for FNH was 44.87 ± 13.3 min, which was significantly lower than the surgical time of 137.6 ± 59.7 min. Shortening the operation time can effectively reduce the risk of bleeding and liver dysfunction. Thermal ablation also showed obvious advantages in anesthesia. In thermal ablation, intravenous sedation and analgesia plus local basic anesthesia were mostly used and could reduce the intake of narcotic drugs and the risk of related damage. Most patients had no obvious pain during the treatment process. In terms of intraoperative blood loss, the amount of intraoperative blood loss in the thermal ablation group was significantly lower than that in the surgical operation group  was >10 cm3 in the thermal ablation group. The postoperative loss of normal liver tissue in the surgical group was significantly greater than that in the thermal ablation group (p < 0.05). The volume loss of normal liver tissue mainly correlates with enlarging the ablation range and the method of surgical resection used. For FNH lesions with a definite diagnosis, there is no need to enlarge the ablation range. Meanwhile, FNH is rich in blood supply; the ablation heat well surrounds the entire lesion; hence, it causes little damage to normal liver tissue. In this study, 92.3% (36/39) belonged to this situation. For lesions of unclear nature, especially those in which malignancy could not be excluded, ablation must be carried out in strict accordance with the ablation standard of the malignant tumor (0.5–1 cm beyond the edge of the lesion) to ensure effective inactivation of the lesion (3 was about 83.7% (36/43), and only seven cases of local liver tumor excision had the least normal liver tissue injury. Although the surgical operation and thermal ablation only need to resect the lesion, due to the complexity of the surgical operation, it is difficult to only resect the lesion. Thermal ablation can also follow and can protect normal liver tissue and ensure stable normal digestive function. In conclusion, thermal ablation is superior to surgery in terms of anesthesia, operation time, intraoperative bleeding, and preservation of normal liver tissue with less surgical trauma.The analysis of the postoperative loss of normal liver tissue between both groups showed that 84% (36/43) of the volume of normal tissue lost was >10 cme lesion . In thise lesion . Nonanate lesion , surgicaIn this study, common complications, such as postoperative infection, pleural effusion, and postoperative severe liver damage, caused by thermal ablation and surgical treatment for FNH were compared and analyzed to evaluate the occurrence of postoperative complications in both groups. This study showed that complications of thermal ablation for FNH were less than those of surgery. There was a significant difference between both groups. In this study, there were four cases  with complications such as severe liver dysfunction and pleural effusion in the thermal ablation group, including two cases of postoperative pleural effusion and two cases of severe liver dysfunction with transaminase >500 U/L. Both of the patients with severe liver dysfunction had multiple lesions, five lesions in one case and three lesions in the other, which were considered to be caused by ablation of multiple lesions. After treatment for liver preservation, the patients returned to normalcy approximately 2 weeks later. In two cases who had pleural effusion, one was caused by intraoperative chest wall hemorrhage, while the other was a very small amount on the right diaphragm, with no special treatment given; the 1-month follow-up examination was self-absorbed. In the surgical group, there were 16 cases  of postoperative complications, including eight cases of pleural effusion, two cases of postoperative infection, and six cases of severe liver dysfunction. Among the eight cases of pleural effusion, two were complicated by pulmonary infection, which mainly correlated with long postoperative time in bed. Severe liver dysfunction occurred in six patients postoperatively, which may correlate with operation time and multiple lesions . Among the two infected patients, one suffered incision infections and was cured by drug treatment, while the other patient had incisional and extra-abdominal cavity infection 5 days after the operation, which occurred in the intracranial and buccal cavity, causing speech impediment and motor dysfunction. It improved after anti-infection therapy and rehabilitative exercise for 3–5 months. The inducement of infection may be related to the patient’s long operation time (approximately 240 min) and large intraoperative blood loss (approximately 500 ml), which may cause greater damage to the body and result in significantly weakened resistance. In conclusion, compared to surgical treatment, thermal ablation of FNH has less intraoperative trauma, resulting in fewer postoperative complications than surgical treatment, and the overall incidence of complications is better than that of surgical treatment. This study analyzed the physical and psychological comfort of patients in the treatment of the two groups by comparing postoperative fasting time, indwelling time of abdominal drainage tubes, scar size, degree and time of postoperative pain, length of hospital stay, and total hospitalization expenses. The results showed that the physical and psychological comfort of patients treated with FNH thermal ablation was significantly better than that of patients treated with surgery. After thermal ablation for FNH, there was no need for a drainage tube , and only 4–8 h of fasting was required under local anesthesia and sedation. The scar was <5 mm, and there was less postoperative hospitalization time and total treatment costs, all of which were beneficial to patients. Small trauma and a comfortable treatment experience can help patients recover quickly. In conclusion, thermal ablation of FNH can give patients a better medical experience and is both economic and practical, with significant advantages over surgical treatment.In recent years, rehabilitation or enhanced recovery programs for liver surgery have been gradually applied to clinical practice. Relevant clinical studies have shown that compared with traditional standardized treatment, preoperative rehabilitation programs and perioperative steroid administration can effectively reduce overall complications , 22. In In conclusion, thermal ablation has less trauma, fewer complications, and is economical and practical. For patients with hepatic FNH ≤5 cm who need active treatment, especially the elderly, children, and those who cannot tolerate surgery, thermal ablation is a safe and effective treatment method worthy of recommendation.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the ethical and scientific review board of Fujian Provincial Hospital. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Participants completed a validated food frequency questionnaire, a physical examination, and biochemical analyses. Linear regression was used to examine the associations of dietary antioxidant micronutrients with HbA1c and estimated insulin sensitivity (eIS) in models adjusted for relevant covariates and stratified by diabetes status. In adults with T1D, we observed higher dietary manganese intake associated with higher eIS in the model adjusted for age, sex, diabetes duration, and total calories. In nondiabetic controls, higher intake of manganese associated with lower HbA1c and higher eIS values that persisted in models adjusted for all relevant covariates. On the other hand, dietary copper revealed a positive association with HbA1c in models adjusted for all covariates, except BMI and plasma lipids. No associations were noted for vitamins C and E and dietary carotenoids in either group. These findings reveal dietary antioxidant micronutrients, especially trace elements such as copper and manganese deserve special attention in glycemic control in adults with T1D as well as in nondiabetic controls.This trial is register with NCT00005754.Type 1 diabetes (T1D) has been associated with increased risks of atherosclerotic cardiovascular disease, and poor glycemic control and oxidative stress play a major role in its pathology. There is a lack of data on the role of dietary antioxidant micronutrients, including vitamins and trace elements, in glycemic control in T1D. The aim of this study is to examine associations of dietary intakes of micronutrients with glycemic status. We report data from a cross-sectional analysis from the coronary artery calcification in type 1 diabetes (CACTI) study ( Type 1 diabetes (T1D) has been associated with a significant burden of atherosclerotic cardiovascular disease (ASCVD) and subsequent mortality when compared to the general population . OxidatiCross-sectional studies reveal intakes of antioxidant vitamins, especially vitamins C and E that do not meet the dietary recommendations in adults with T1D; however, these studies do not report their associations with glycemic control , 13. UsiThe Coronary Artery Calcification in Type 1 Diabetes (CACTI) is a prospective study examining the progression of ASCVD and its association with dietary, lifestyle, and biochemical factors in adults with T1D compared to matched nondiabetic controls. We have previously reported altered macronutrient intakes, especially higher dietary fats from animal sources , and dieNCT00005754).Data presented in this report were collected at baseline of the CACTI study as previously reported . All stuDetailed dietary data were obtained using a food frequency questionnaire Harvard 1988) as previously described . Baselin88 as pret test for continuous variables and χ2 test for categorical risk factors. In addition, Wilcoxon rank sum test was used to analyze variables with a skewed distribution. Factor analysis was used to examine dietary patterns, and foods were considered to form part of the dietary patterns with a load factor greater than 0.4. A multiple linear regression model was used to examine associations of dietary antioxidant micronutrients with HbA1c and eIS as clinical markers of glycemic control. Models were adjusted for relevant covariates as follows: model 1 , model 2 (model 1 + BMI), model 3 (model 1 + plasma LDL-cholesterol and triglycerides), and model 4 (model 1 + dietary patterns and food groups). These models were stratified by diabetes as we observed significant interaction effects. All P values were 2-tailed, and main effects and interaction effects were considered if P was ≤0.05. Analyses were performed using SAS version 9.4 .Differences in baseline characteristics were analyzed between adults with and without T1D using Student Adults with T1D were younger in age and had a greater proportion of females than the nondiabetic controls. As expected, T1D adults had higher serum HbA1c and lower eIS than the nondiabetic controls. Lipid profiles were lower in the T1D group (LDL-cholesterol and triglycerides). No significant differences were noted in BMI and total caloric intake between the two groups. Among the dietary intake of antioxidant micronutrients, vitamins C and E, as well as iron, copper, zinc, selenium, manganese, and carotenoids, did not differ between the two groups .In a multivariate model adjusting for relevant covariates including dietary patterns and food groups, no associations were observed between any of the dietary antioxidant micronutrients and serum HbA1c in adults with T1D. On the other hand, in nondiabetic controls, dietary copper revealed a significant positive association with HbA1c in models adjusted for age, sex, total calories, dietary patterns, and food groups. We also observed a significant inverse association of dietary manganese intake with HbA1c in nondiabetic controls persisting in all adjusted models .In adults with T1D, dietary intake of manganese revealed a significant positive association with insulin sensitivity only in the minimally adjusted model. On the other hand, in nondiabetic controls, dietary manganese intake was consistently associated with higher insulin sensitivity in all adjusted models. Finally, we also observed a borderline significant inverse association of dietary copper intake with insulin sensitivity in non-diabetic controls .Overall, this cross-sectional analysis shows a positive association of dietary manganese with insulin sensitivity that remained significant in all adjusted models in adults without diabetes and remained significant in minimally adjusted model in T1D group. We also observed higher intake of copper associated with higher HbA1c levels in nondiabetic controls, thus indicating poor glycemic control; this association remained significant in models adjusted for all dietary patterns as well as age, sex, and total calories. Finally, we also observed a consistent protective (inverse) association of the trace element manganese with HbA1c that remained significant in all adjusted models in nondiabetic controls.Dietary copper is a redox active metal and has been shown to cause impaired glucose metabolism in mechanistic and observational studies –25. Our In contrast to dietary copper, dietary manganese revealed a protective association against higher HbA1c and promoted insulin sensitivity in our cohort. Dietary manganese intake has been inversely associated with the development of type 2 diabetes, as well as with biomarkers of inflammation in multiple prospective cohort studies –32. Our Our observation of no association of dietary vitamins C and E with glycemic control conforms to the findings of meta-analyses which showed no beneficial effects of their supplementation in glycemic control and insulin resistance in adults with or without diabetes , 36, 37.Our study has some limitations as follows: Our cross-sectional analysis cannot address causality, and we did not measure serum/tissue levels of these antioxidant micronutrients which could shed greater light on these observed associations. Being limited to adults with established ASCVD and their matched nondiabetic controls, our findings cannot be generalized to other populations, namely, those with type 2 diabetes or gestational diabetes or younger adults with T1D but without ASCVD. Furthermore, in the current report, we analyzed dietary sources of these micronutrients and not those derived from dietary supplements, and their bioavailability that must be addressed in future studies. Finally, there is always the possibility of residual confounding in epidemiological studies that cannot be addressed in the analysis. Despite these limitations, the major strength of our study design is a well-characterized cohort of adults with T1D and their matched controls providing a clear comparison of associations between these two groups. Also, our significant findings remained robust in models extensively adjusted for major dietary patterns and food groups.Dietary antioxidant nutrients, including vitamins and trace elements, play a major role in regulating antioxidant/oxidative stress pathways that have close implications in insulin signaling and glycemic control. Based on data collected from dietary intakes, and biochemical variables of glycated hemoglobin and insulin sensitivity, we examined associations of these factors in our cross-sectional observational study. Our findings reveal the importance of dietary copper and manganese in glycemic control in adults with type 1 diabetes, as well as in nondiabetic controls. Dietary copper may promote poor glycemic control, while manganese could be protective against hyperglycemia and promote insulin sensitivity. These nutrients and their food sources deserve attention in the nutrition therapy for diabetes and overall blood glucose control.


The synthesis of nanoparticles from noble metals has received high attention from researchers due to their unique properties and their wide range of applications. Silver nanoparticles (AgNPs), in particular, show a remarkable inhibitory effect against microorganisms and viruses. Various methods have been developed to obtain AgNPs, however the stability of such nanostructures over time is still challenging. Researchers attempt to obtain particular shapes and sizes in order to tailor AgNPs properties for specific areas, such as biochemistry, biology, agriculture, electronics, medicine, and industry. The aim of this study was to design AgNPs with improved antimicrobial characteristics and stability. Two different wet chemical routes were considered: synthesis being performed (i) reduction method at room temperatures and (ii) solvothermal method at high temperature. Here, we show that the antimicrobial properties of the obtained AgNPs, are influenced by their synthesis route, which impact on the size and shape of the structures. This work analyses and compares the antimicrobial properties of the obtained AgNPs, based on their structure, sizes and morphologies which are influenced, in turn, not only by the type or quantities of precursors used but also by the temperature of the reaction. Generally, AgNPs obtained by solvothermal, at raised temperature, registered better antimicrobial activity as compared to NPs obtained by reduction method at room temperature. Silver nanoparticles (AgNPs) are currently used in various fields, as medicine , biologyAgNPs are well known for possessing an inhibitory effect to the most common microorganisms in the medical and industrial processes ,22. AgNPThere are several methods used in the synthesis of silver nanoparticles such as biological ,26, physThe chemical reduction method has been used the widest studied, due to the general versatility of the technique ,35,36. CThe advantage of the chemical synthesis of nanoparticles is related to the ease of production, low cost, relatively short time of reaction and high yield ,40,41. BThe formation mechanism consists in the reduction of silver ions into free metallic silver atoms which are further lead to the formation of the nanoparticles (by growing), in a bottom–up approach. The chemical reduction consists by the addition of reducing agents as  which lead to form of free metallic silver atoms. As the reaction proceeded, under the influence of reaction conditions, such as magnetic stirring, temperature, pH, these silver atoms accumulated into oligomer clusters and finally these clusters lead to the formation of silver colloids ,45. + [The present work proposes two synthesis methods (i) chemical reduction at the room temperature using reducing agents, such as sodium borohydride at different concentrations and tri-sodium citrate. The stabilizing agent polyvynilpirrolidone (PVP) has a role to prevent the agglomeration of nanoparticles during synthesis or deposit them . It was + ,49. (ii)+ ,51,52,53The chemicals products have analytical grade and were used without further purification. The water used is deionized 18 MΩ. The silver nitrate is from Silal Trading, Bucharest, Romania (99.999%), the sodium borohydride is from Sigma Aldrich Co., Ltd. , the tri-sodium citrate dihydrate is from Merck Co., Ltd.  and PVP K30 is from Roth Co., Ltd. . −2 M) solution mixed vigorously (600–700 rpm) after 10 min was added 30 mL of PVP solution 0.7 mM and various volumes of sodium borohydride. The mixture was magnetic stirring for 2–3 min, at room temperature then added 1–3 mL hydrogen peroxide 35% (A solution of silver nitrate (0.1 mM) was prepared in 500 mL deionized water in a 1000 mL flask, then was added 30 mL tri-sodium citrate dihydrate  respectively their molar concentration  with an average molecular mass of 400 (Aldrich), analytical purity. Deionized water with resistance of 18 MΩ was used. The synthesis of uniform silver nanospheres using solvothermal way  were fol mixture . The obt mixture  and at c mixture . The con mixture 2 F30 S-TWIN high resolution transmission electron microscope  operated at 300 kV. The TEM samples were prepared as follows: a small amount of sample was diluted in deionized water. Then 10 µL of the diluted solution it was placed onto a 400-mesh holey carbon coated Cu grid and let it to dry in air prior to the analysis. The UV–Vis spectra were recorded using an Evolution 300 UV-VIS spectrophotometer in absorbance mode (190–1100 nm), 1 nm used as data interval, 2 nm bandwidth and 240 nm/min scan speed, in 10 mm quartz cuvettes, at room temperature. The DLS measurements were done using a DELSA Max Pro, light scattering analyzer reusable PEEK flow cells, BCI-3216-DMP, DLS detector angle (degree) 163.5°. The acquisition parameters for this technique were: acq time (s): 5, read interval(s): 1, number acq: 3, electric field frequency (Hz): 10, collection period (s): 15, auto-attenuation: yes, attenuation level (%): 0, laser mode: normal, set temp on connection: no, set temp (°C): 20. The dilution factor used was 10 (1 mL in 10 mL of water). TEM images were acquired using a by Tecnai GMicrobial strains were purchased from American Type Culture Collection . Glycerol stocks were streaked on LB agar to obtain 24 h cultures to be used for all further studies.Staphylococcus aureus ATCC® 23235 and Enterococcus faecalis ATCC® 29212) and Gram-negative (Escherichia coli ATCC® 25922 and Pseudomonas aeruginosa ATCC® 27853) groups and also a yeast model, Candida albicans ATCC 10231. Briefly, 0.5 McFarland (1–3 × 108 CFU (colony forming units)/mL) suspensions in sterile saline were obtained from overnight cultures, previously cultivated in nutritive agar. The obtained microbial suspensions were utilized to swab inoculate Mueller Hinton agar Petri dishes, as for disc diffusion technique described in CLSI 2020 standard  [To qualitatively assess the antimicrobial potential of obtained nanoparticles, an adapted diffusion method in nutritive agar was performed. We used 4 bacterial strains models, belonging to Gram-positive (er 2020) . Then, 18 CFU/mL) were added in each well. The MIC values were established by naked eye analysis and spectrophotometric measurement (Abs 600 nm). Each experiment was performed in triplicate and repeated on at least three separate occasions.For establishing the MIC (minimum inhibitory concentration) values of the obtained functionalized silver nanoparticles we utilized a microdilution method performed in nutritive broth. The sterile broth was added in sterile 96 well plates and binary dilutions of each tested compound were performed in a final volume of 150 μL. After realizing the binary dilutions, 15 μL of microbial suspension adjusted to an optical density of 0.5 McFarland  was gently removed. The obtained biofilms attached on the 96 well plate’s walls were washed with sterile saline buffer and then biofilms were fixed with cold methanol for 5 min. After fixation methanol was removed and air-dried plates were stained with 1% crystal violet solution for 15 min. After staining plates were washed and allowed to dry at room temperature. Biofilm formation ability was assessed by adding 150 µL of 33% acetic acid solution to release the dye included in the biofilm cells. The intensity of the resulting solution (reflecting the intensity of biofilm development) was measured with a spectrophotometer (Abs 490 nm).To assess the effect of tested nanomaterials on the biofilm formation of tested microbial strains, experiment was set up in 96 well plates, in a similar fashion as described in MIC evaluation protocol. Microbial cells adjusted at a 10The optical and structural properties and the stability of the obtained samples were evaluated by UV-VIS spectroscopy. UV-Visible spectroscopy is one of the most widely used techniques for structural characterization of silver nanoparticles . The tecThe obtained silver nanoparticle solutions at room temperature named RT are different in colors  and showThe samples obtained by chemical reduction at room temperature encoded RT show peaks around the value of the wavelength of 320 nm indicating the ions silver presence  and peakThe spectra show a shifting of the absorption band for samples encoded with RT1 and RT2 prepared with the lesser and highest amount of the strong reducing agent .4) show a small peak around values 460–490 nm which indicate probably, the presence of some spherical shapes of silver nanoparticles , drive to the triangular shapes nanoparticles. The PVP agent is acting also as a directing agent, in this case, confirmed by the color sample (violet color) with a slight agglomeration tendency confirmed by UV-VIS data. In this case using various amount of the strong reducing agent, stabilizing agent and silver precursor the shape, size and the stability of the final colloidal solutions are tuned.Based on UV-VIS data, the chemical reduction proposed by varying the ratio of reduction agents lead to the control of sizes and shapes of nanoparticles. The UV-VIS spectra  indicate4) as reducing agent in the chemical reduction synthesis led to silver nanoparticles with specific sizes and shapes indicated by the color of obtained solution. The blue dark or lighter color is a specific for chemical reduction using sodium borohydride and indicate anisotropic shapes. Pal et al. [Using the sodium borohydride  . Thus, fThe values mentioned in the 4) (sample coded with RT2) driven to the heterogeneity of size distribution  but smaller value of the polydispersity  comparinribution . Compariribution  it is noAccording to the DLS obtained results after 8 months from the first test it can be seen all samples have multimodal dispersity . All samTEM results for samples RT1 and RT3 show a relative homogeneous distribution confirmed by corresponding DLS data . The sphBased on TEM results from RT1 and RT3 particle sizes were measured with Image J software. The obtained values were plotted using OriginPro2021 and statistically evaluated for average size and standard deviation measurements. From the obtained values the error was calculated. Thus, for these measurements, 250 nanoparticles from different areas were taken into consideration, for each sample.4) added at the sample RT1 could influence the kinetic reaction for nanoparticles formation led to obtaining oval or spheres shapes  have different colors from deep yellow color to dark brown depending on the temperature and time applied during the solvothermal synthesis.UV-VIS spectra  for the UV-VIS spectra  for all DLS analysis were done in special cuvettes with diluted solution. The DLS images from samples obtained by solvothermal method  show by The graphic images from DLS graphic  of samplBased on TEM results from HT1 and HT4 particle sizes were measured with Image J software. The obtained values were plotted using OriginPro2021 and statistically evaluated for average size and standard deviation measurements. From the obtained values the error was calculated. Thus, for these measurements, 250 nanoparticles from different areas were taken into consideration, for each sample.TEM images of samples obtained by the solvothermal method encoded with HT1 and HT4 at different resolutions showed some morphological differences in the obtained nanoparticles. The AgNPs sample coded HT1 treated at 200 °C  show a uThe sample HT4 b indicatAnalyzing diameters distribution for samples encoded HT1c and HT4S. aureus) and Gram negative  bacteria, but also against the opportunistic yeast C. albicans. HT1NPs and HT4NPs provided diameter higher diameter of inhibition zones, ranging 8–12 nm in all the test situations  and quantitative (MIC) experiments in both planktonic and biofilm cultures ,39,70,71tuations . CompariHT samples showed spherical shapes of nanoparticles formation indicated by the TEM images  and haveS. aureus as compared to their antimicrobial effect on the other microbial strains. However, solvothermal samples (HT) clearly show enhanced antibacterial effect against S. aureus, with average diameter of inhibition zones of 11.5 cm, while growth inhibition diameter averages 7 cm for RT samples in this Gram-positive bacteria.Comparing the RT samples with samples obtained by solvothermal synthesis (HT) ones it is noticed that RT samples obtained by chemical reduction at room temperature show better efficiency relatively better against gram-positive bacteria +) could bind to active sites of the S. aureus disrupting the bacteria cell by thiol-redox homeostasis and increased ROS production, which induced bacteria death finally [It is noticeable that the samples delivered from the synthesis at the room temperature has the efficiency for Gram-positive bacteria. This is might be possible the less concentration of AgNPs could affect the antimicrobial efficiency for the Gram –negative bacteria while for Gram-positive bacteria the silver ions  is a quantitative method, based on obtaining a serial binary microdilution in liquid nutritive media. The graphic in P. aeruginosa strain . Lowest MIC values obtained for HT samples ranging 0.03–0.06 mg/mL, as revealed by figure . For solbacteria ,50.E. coli, results showed that biofilms are slightly more efficiently inhibited by the solvothermal nanoparticles encoded HT, as compared to the other samples  and solvothermal method at high temperature (HT). The results showed that AgNPs obtained by the room temperature (RT) method registered a various morphology with truncated or bipyramidal aspect and the sizes around 1–20 nm. Samples obtained at HT with spherical shapes and size of nanoparticles more than 50 nm have relatively better antimicrobial activity than AgNPs coded with RT. Also, these present a lower agglomeration tendency than the nanoparticles obtained at the room temperature. These differences could be related to the condition of the medium reaction utilized during synthesis which driven to a variation of shapes and sizes. AgNPs obtained at room temperature are the easiest method for obtaining nanoparticles but, shapes, sizes and stability in time are highly influenced by environmental conditions. The solvothermal method is a more expensive method that requires careful monitoring of the reaction that takes place in special conditions. Due to the special conditions in which the reaction takes place, at constant pressure, and high temperatures the AgNPs obtained have sizes range higher compared with the chemical reduction at room temperature, and are more stable and have uniform shapes than the samples obtained at the room temperature. Even if the synthesis at room temperature is easier to achieve, the variables during reaction can lead to different morphologies of nanoparticles and greater tendency towards agglomeration. The antimicrobial activity of the developed NPs depends on the method type, chemicals and synthesis conditions. Samples obtained at high temperature method showed the highest inhibitory effects, being active on a wide range of Gram positive, Gram negative, and yeast strains, which are relevant for human pathology. The obtained AgNPs showed low minimum growth inhibitory concentrations and biofilm inhibitory concentrations, usually ranging 0.03–0.12 mg/mL, values being higher for biofilms, as expected. Comparing both syntheses The results of both syntheses proved once again the significance of the followed method depending on the specific reaction parameters which influence the final morphology of nanoparticles, the physico-chemical and biological properties of the nanoparticles of the noble metals.


The embryonic development of the human umbilical cord (hUC) is complex, and different regions can be identified in this structure. The aim of this work is to characterize the hUC at in situ and ex vivo levels to stablish their potential use in vascular regeneration. Human umbilical cords were obtained and histologically prepared for in the situ analysis of four hUC regions , and primary cell cultures of mesenchymal stem cells (hUC-MSC) isolated from each region were obtained. The results confirmed the heterogeneity of the hUC, with the IV and PV zones tending to show the higher in situ expression of several components of the extracellular matrix , vimentin, and MSC markers , although isolation and ex vivo culture resulted in a homogeneous cell profile. Three vascular markers were positive in situ, especially vWF, followed by CD34 and CD31, and isolation and culture revealed that the region associated with the highest expression of vascular markers was IV, followed by PV. These results confirm the heterogeneity of the hUC and the need for selecting cells from specific regions of the hUC for particular applications in tissue engineering. The human umbilical cord (hUC) is a conduit about 50–60 cm long and 1–2 cm in diameter, connecting the abdominal wall of the fetus to the placenta for exchange of nutrients and gasses during gestation .The hUC is a heterogeneous structure in which several cell subpopulations and ECM configurations can be found . AlthougCells isolated from the hUC demonstrated a high expansion potential, plasticity, and immunomodulatory properties, and have been proposed as potentially useful in stem cell therapies ,9. In faIn the present work, we have carried out a histological, histochemical, and immunohistochemical analysis of the hUC cells and ECM in order to evaluate the profile of each region of the hUC as a source of cells for use in vascular tissue engineering.With the consent of the parents, five human umbilical cords (hUCs) were obtained from full-term newborns delivered by previously-scheduled cesarean section due to obstetric reasons. Samples were washed in 1× phosphate buffered saline (PBS) and cut into 1.5 cm-length pieces. For histological analysis, transversal sections of the five hUCs were fixed in 4% formaldehyde for 48 h at room temperature, washed, dehydrated, cleared, and embedded in paraffin following standard histological protocols. Tissue sections were obtained, dewaxed, and stained with hematoxylin and eosin (HE) and examined with a Nikon Eclipse 90i light microscope . For scanning electron microscopy (SEM), samples were fixed in 2.5% buffered glutaraldehyde in 0.05 M cacodylate buffer at 4 °C overnight and then washed three times in the same buffer at 4 °C. Fixed samples were dehydrated in increasing concentrations of acetone  and completely dried using the critical point drying method. All these reagents were obtained from Panreac Química S.L.U., Barcelona, Spain. Dried samples were mounted on aluminum stubs, covered with gold-palladium and examined with a FEI Quanta 200 scanning electron microscope using the high vacuum mode .Analysis of relevant ECM components of the hUC was performed by histochemistry. First, collagen fibers were identified by using Picrosirius red (PR) methods. Briefly, tissue sections were stained with a Sirius red F3B working solution for 30 min and counterstained with Harris Hematoxylin for 5 min. For proteoglycans detection, alcian blue (AB) was used by staining dewaxed sections in the AB reagent solution for 30 min, followed by PBS washing. To detect ECM glycosaminoglycans, sections were stained with periodic acid-Schiff (PAS). In this case, tissues were incubated in a 0.5% periodic acid solution for 5 min as an oxidant, followed by incubation in Schiff reagent for 15 min and counterstaining with Harry’s hematoxylin for 1 min . Normal human tissues were used as technical controls for each method.Clostridium histolyticum type I collagenase . Isolated cells were collected by centrifugation and seeded in 75 cm2 culture flasks  using Amniomax-C100 culture medium (Gibco-Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics . Cells were kept using standard cell culture conditions at 37 °C in a humidified incubator with 5% CO2 to generate cultured human umbilical cord mesenchymal stem cells (hUC-MSC). Once the cells reached 70% confluence, they were trypsinized with 0.25% trypsin-ethylenediaminetetraacetic acid -EDTA- (Merck). All hUC-MSC were used at cell passages 1 to 3.For the ex vivo analysis, hUC biopsies were carefully analyzed using a stereo microscope, and biopsies corresponding to each topographical region of the hUC  were obtained using a 1mm surgical punch. Once pre-dissected hUC zones were obtained, tissue sections were digested for 6h at 37 °C in a 2 mg/mL solution of 5 cells of each zone were placed in flow cytometry tubes and washed with 2mL of staining buffer . Then, the Fc receptors were blocked by incubating the cells for 5 min with 2 mL of PBS containing 0.1% bovine serum albumin and 0.1% FBS. Next, cells were stained with a positive cocktail of markers  and a negative cocktail  and incubated for 45 min at 4 °C in darkness. Once cells were labeled, the cells were placed in a staining buffer and analyzed using a FACSCalibur flow cytometer (BD Biosciences), and the percentage of positive cells was quantified in each study group.hUC-MSC isolated from each region were analyzed by flow cytometry to determine the expression of typical MSCs markers using a human MSC Analysis BD Stemflow™ kit . In brief, 5 × 102O2 to quench endogenous peroxidase. Each of the succeeding steps was followed by a thorough rinse in 1× PBS and were performed in a humid chamber to prevent tissue desiccation. Non-specific staining was blocked by incubation in 2.5% horse serum and casein . Then, samples were incubated overnight with primary antibodies at 4 °C. After that, samples were incubated with secondary antibodies at room temperature for 1h. The complex epitope antibody was detected using 3,3-diaminobenzidine -DAB- (Vector Laboratories), and tissue sections were counterstained for 20 s using Harry’s hematoxylin (Gibco-Thermo Fisher Scientific). Human control tissues were used as technical controls. In all cases, images were obtained using a Pannoramic® DESK II DW scanner . The antibodies used and technical details of the immunohistochemical procedure are summarized in Analysis of relevant stromal and MSC markers , ECM components , and vascular cell markers  was performed by immunohistochemistry. As detailed in 4 cells were cultured in chamber slides , fixed with 70% ethanol, washed in 1× PBS, and blocked for 30 min with normal horse serum and casein (Vector Laboratories). Then, samples were incubated for 1 h at room temperature with primary antibodies. Subsequently, samples were washed in 1× PBS and incubated for 1 h with FITC-conjugated anti-mouse secondary antibodies (Merck). The technical details of the immunofluorescent procedure are summarized in Relevant MSC markers were analyzed by immunofluorescence in cultured hUC-MSC. Briefly, 1 × 10n = 15).In the first place, the results obtained for the histochemical analyses  and for the immunohistochemical analysis of ECM components  were quantified as previously described . In briep < 0.05 were considered statistically significant in a two-tailed test. Statistical testing was carried out using the Real Statistics Resource Pack software (Release 7.2) available at https://www.real-statistics.com/  (accessed on 15 April 2022).Averages and standard deviations were calculated for each sample and each analytical method, and comparison between groups was performed using the Mann–Whitney test, since distributions demonstrated to be non-normally distributed, according to the Shapiro–Wilk test. Values of The in situ histological analysis of the hUC stained with HE confirmed the presence of and external layer composed of amnioblast cells, three inner vascular structures , and a mucous connective tissue A. This mThe analysis of relevant ECM components in the hUC sections using histochemistry revealed several differences among the hUC regions . First, The characterization of relevant MSC markers in hUC cells was determined by immunohistochemistry for CD73, CD90, and CD105 markers. In the first place, the analysis in situ of hUC sections  revealedNext, we evaluated the expression of the same MSC markers in the hUC-MSC isolated from the cord and kept in ex vivo culture using flow cytometry. The results of this analysis  showed tOn the one hand, in situ analysis of VIM expression, an intermediate filament protein expressed by mesenchymal stem cells, showed that all cells found at the four hUC zones  were positive for this marker . On the The analysis of three vascular markers showed that hUC cells intrinsically expressed some of these markers in situ in the hUC . For CD3Although it is well known that the hUC contains diverse zones with substantial histological differences in their ECM matrix and cell profile ,5,6, theThese results could be related to the proximity of IV and PV to the umbilical blood vessels and the high pressures that must be withstood by these structures ,21. FurtThese structural differences detected by histology and histochemical assays led us to study the stemness profile of cells allocated at each hUC region. It has been previously established that cells used in tissue engineering and cell therapy should meet several criteria established by the International Society for Cellular Therapy (ISCT) for cells . These iOn the other hand, we carried out several immunohistochemical analyses to identify several key components of the hUC. In the first place, we confirmed that all cells were positive for vimentin, both in situ and after ex vivo isolation. As a stromal marker playing an important role in maintaining cell integrity, the presence of vimentin confirms the stromal nature of these cells ,26. In tFinally, we analyzed three key markers of vascular cell differentiation in the hUC cells to determine the potential usefulness of these cells in vascular tissue engineering. The identification of an adequate source of cells with vascular potential could be very useful for the generation of large vascular structures resembling arteries and veins ,28, but In this regard, we found that CD31, a protein with an important role in endothelial cell adhesion . HoweverThe present study has some drawbacks and limitations that should be addressed in future research. First, independent studies should evaluate the multilineage mesodermal differentiation potential of each hUC zone. Second, the transitional zones allocated among the different hUC zones should be studied to clarify the differences between hUC zones. Furthermore, long-term studies should be focused on the identification of changes in the expression of vascular markers in subsequent subcultures of cells derived from the four hUC zones to determine the most suitable cell passage for tissue engineering applications. Finally, our results were obtained using a small group of samples. Therefore, results should be validated in a larger cohort of cords to assess intersample variability.In summary, our results confirm the heterogeneity of the different regions of the hUC and the need for selecting specific regions for tissue engineering purposes. Although cells showed different profiles in situ, we found that the isolation and culture of hUC-MSC corresponding to different regions resulted in a very similar profile of cultured cells for some parameters, but specific differences were found for the vascular markers CD31, CD34, and vWF, suggesting that cells corresponding to IV should be preferentially isolated for vascular tissue engineering. In addition, the high potential of cells isolated from the umbilical cord suggests that these cells could also be used for the generation of other human organs and tissues such as the human skin, cornea, and oral mucosa.


B. subtilis pectinase was immobilized on iron oxide nanocarrier. Through co-precipitation, magnetic iron oxide nanoparticles were synthesized. Scanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEX) were used to analyze the nanoparticles. Pectinase was immobilized using glutaraldehyde as a crosslinking agent on iron oxide nanocarrier. In comparison to free pectinase, immobilized pectinase demonstrated higher enzymatic activity at a variety of temperatures and pH levels. Immobilization also boosted pectinase’s catalytic stability. After 120 h of pre-incubation at 50 °C, immobilized pectinase maintained more than 90% of its initial activity due to the iron oxide nanocarrier, which improved the thermal stability of pectinase at various temperatures. Following 15 repetitions of enzymatic reactions, immobilized pectinase still exhibited 90% of its initial activity. According to the results, pectinase’s catalytic capabilities were enhanced by its immobilization on iron oxide nanocarrier, making it economically suitable for industrial use.To increase its operational stability and ongoing reusability,  Pectinase is a group of heterogeneous enzymatic reactions for the degradation of pectin and pectin-containing compounds. Pectinases are used in a variety of industrial processes, including fruit juice extraction, textile processing and bioscouring of cotton fibers, degumming of remain, retting of flax, coffee and tea fermentation, oil extraction, plant protoplast formation, and pretreatment of pectic waste during water treatment ,2,3. In There are several uses for immobilized biocatalysts in industrial and commercial processes. These techniques provide greater operational cycle control, resulting in a more flexible reactor design. The separation of immobilized biocatalysts from the process is a simple and cost-effective method for downstream product purification .There are numerous methods for immobilizing enzymes. Each method has its own benefits and drawbacks. By optimizing several circumstances for a given enzyme, it is possible to select an appropriate technique. Immobilization techniques include adsorption, entrapment, covalent bonding, and cross-linking. The adsorption and entrapment processes are easier, but weak contacts cause enzyme leaching. However, covalent binding and cross-linking produce strong interactions, but they can alter the shape of enzyme structures, resulting in enzyme inactivation . In the There have been reports of the immobilization of pectinase using a variety of support materials, including alginate beads , agar geDue to their chemical, physical, and superparamagnetic properties, as well as their high surface area for reactivity, magnetic nanoparticles (MNPs) have been deemed promising carriers for immobilizing enzymes ,18,19. T+3/Fe+2 ratio. Equation (1) describes the reaction process in detail.The nanoparticles were synthesized using the co-precipitation process. This is the simplest and most efficient approach for synthesizing metal oxide nanoparticles. In this approach, stoichiometric quantities of metal salts in proper molar ratios are introduced to basic media (pH 8–14), resulting in precipitate formation. The magnetic nanoparticles were synthesized using a 2:1 Fe3O4 is prone to oxidation and is extremely unstable, it is further oxidized in the presence of air through the calcination process, resulting in the formation of stable γ-Fe2O3 (Equation (2)).As FeScanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEM) were used to characterize the nanoparticles that were produced (EDAX). The particle size and surface morphology of iron oxide nanoparticles were investigated using SEM. The electron micrograph of iron nanoparticles is shown in Glutaraldehyde often crosslinks the enzyme through intra- and intermolecular cross-linking formation reactions. While a low dosage of glutaraldehyde may not be sufficient to bind a maximum concentration of enzyme with maximum enzymatic activity, a high concentration of the crosslinking agent may alter the structure of enzymes, rendering them inert. As a result, the impact of glutaraldehyde concentration on pectinase immobilization in terms of maximal enzymatic retention was investigated using different concentrations of glutaraldehyde ranging from 0.5% to 5.0%. . The resThe optimal enzyme concentration is critical for retaining maximum residual activity following immobilization. During the immobilization technique, the concentration of pectinase was optimized for optimal residual activity by utilizing several concentrations of enzyme solution ranging from 500 to 5000 IU/mg. The residual activity of pectinase immobilized magnetic iron oxide nanocarrier increased with increasing enzyme concentration, with 2500 IU/mg exhibiting the highest residual activity. The residual activity of immobilized pectinase achieved saturation at a concentration of 2500 IU/mg, and the residual activity of nanocarrier immobilized pectinase remained unchanged when the enzyme concentration was raised further .The impact of crosslinking duration on pectinase immobilization on nanocarrier was investigated by reacting glutaraldehyde solution with nanocarrier for various time periods ranging from 30 to 300 min. The maximal residual activity of immobilized pectinase was observed after 150 min of reaction time, and after this time period, the residual activity of nanocarrier-immobilized pectinase decreased . The shoThe impact of temperature on pectinase immobilization on magnetic iron oxide nanocarrier was investigated by performing the immobilization technique at various temperatures ranging from 5 °C to 40 °C. The immobilization yield of nanocarrier-immobilized pectinase increased with temperature, with the maximum immobilization yield achieved at 30 °C. The immobilization yield of pectinase was reduced as the temperature increase .The effect of incubation time on the enzymatic response of immobilized pectinase was studied by running the enzyme for different time periods ranging from 5.0 to 60 min and comparing it to free pectinase. Immobilization had no effect on the response of time pectinase, and both nanocarrier-immobilized pectinase and free pectinase exhibited maximal relative activity after 10 min of reaction time . The magThe determination of the optimum temperature for maximum enzymatic activity is a critical step in investigating enzyme industrial feasibility; thus, the effect of temperature on the catalytic performance of iron oxide nanocarrier was determined by measuring the enzyme assay at different temperatures ranging from 20 to 60 °C with a comparison of free pectinase . The immThe influence of pH on the catalytic activity of magnetic iron oxide nanocarrier immobilized pectinase was studied by performing the enzyme assay at different reaction pHs spanning from pH 5 to pH 10 and comparing it to free pectinase . The immThe thermal stability of magnetic iron oxide nanocarrier-immobilized pectinase against different temperatures was investigated by pre-incubating the immobilized and free pectinase at various temperatures ranging from 25 °C to 60 °C and measuring enzyme activity every 24 h . It was m and Vmax were calculated by repeating the enzyme assay under standard assay conditions with varied concentrations of citrus pectin. The Km and Vmax values were determined by plotting 1/[V] versus 1/[S] , Sigma , Scharleu , and Merck . All glassware was cleaned by soaking it in detergent and then scouring it with a bristle brush. Before use, it was thoroughly washed with tap water, rinsed with deionized water, and oven dried.Bacillus subtilis, a previously obtained bacterial strain, was employed for pectinase synthesis using the submerged fermentation technique. The pectinase was partially purified from culture media by centrifugation at 10,000× g for 15 min at 4 °C, followed by precipitation with ammonium sulfate at 50% saturation. The precipitates were separated by centrifugation at 10,000 rpm for 15 min, then re-dissolved in glycine-NaOH and dialyzed for 3 h against the same buffer. For immobilization and catalytic characterization, dialyzed pectinase was used.3·6H2O) and ferric chloride hexahydrate (FeCl3·6H2O) were combined in a 1:2 molar ratio  versus 1/[S]  .The reusability of magnetic iron oxide nanocarrier immobilized pectinase was determined under standard assay conditions using citrus pectin as substrate in 15 repetitive cycles and after the magnetic iron oxide nanocarrier, immobilized pectinase was washed with denoised water, and fresh substrate was added for next reaction cycle.Pectinase was generated from B. subtilis and immobilized on magnetic iron oxide nanoparticles using glutaraldehyde as a crosslinking agent in the current study to improve its industrial uses and operational stability. Magnetic iron oxide nanoparticles were prepared using the co-precipitation process and analyzed using scanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEM) (EDAX). When compared to free pectinase, magnetic iron oxide nanocarrier improved pectinase catalytic properties, and immobilized pectinase demonstrated higher relative activity against pH and temperature variations. The thermal stability of pectinase was increased after immobilization of iron oxide nanocarrier, and immobilized pectinase retained more than 90% of its original activity at 50 °C after 120 h of pre-incubation. After 10 batches of reaction cycles, the magnetic iron oxide nanocarrier immobilized pectinase and preserved 100% of its original activity. The immobilization of pectinase in magnetic iron oxide nanocarrier using glutaraldehyde as a crosslinking agent improved its operational stability and catalytic characteristics.


Is there an association between emergency department (ED) use before pregnancy and risk of severe maternal and neonatal morbidity?In this cohort study of more than 2 million births, outcomes were compared between 218 011 patients with an ED visit 90 days before the start of a pregnancy with 1 912 234 without an ED visit. Prepregnancy ED use was associated with a higher risk of severe maternal morbidity, severe neonatal morbidity, stillbirth, and neonatal death, especially as the number of ED visits increased.These findings suggest that ED use may offer an early alert of a pregnant person’s higher future risk of severe adverse maternal and perinatal outcomes. This cohort study examines the association between prepregnancy emergency department use and adverse maternal and perinatal outcomes. Emergency department (ED) use during pregnancy may be associated with worse obstetrical outcomes, possibly because of differences in access to health care. It is not known whether ED use before pregnancy is associated with serious adverse maternal and perinatal outcomes.To study the association between prepregnancy ED use and adverse maternal and perinatal outcomes.This population-based cohort study was conducted in Ontario, Canada, and included all livebirths and stillbirths from April 2003 to January 2020.Main exposure was any ED encounter within 90 days preceding the start of the index pregnancy.Primary outcome was a composite of severe maternal morbidity (SMM) from 20 weeks’ gestation to 42 days’ post partum. Secondary outcomes included severe neonatal morbidity (SNM) from 0 to 27 days, neonatal death, and stillbirth. Relative risks (RRs) were adjusted for maternal age, income, and rurality.Of 2 130 245 births, there were 2 119 335 livebirths (99.5%) and 10 910 stillbirths (0.5%). The mean (SD) maternal age was 29.6 (5.4) years, 212 478 (9.9%) were rural dwelling, and 498 219 (23%) had 3 or more comorbidities. Among all births, 218 011 (9.7%) had a prepregnancy ED visit. The rate of SMM was higher among women with a prepregnancy ED visit than those without (22.3 vs 16.5 per 1000 births), with an RR of 1.34  and an adjusted RR (aRR) of 1.37 . Compared with no prepregnancy ED visit, the aRR was higher in those with 1 , 2 , and 3 or more  ED visits. Prepregnancy ED visits for a hematological , endocrine , and circulatory  conditions were associated with the highest aRRs for SMM. The rate of SNM was higher among newborns whose mother visited the ED within 90 days before pregnancy  as was the risk of neonatal death  and stillbirth .In this study, ED use was common before pregnancy. These findings suggest that ED use may not only reflect a woman’s access to prepregnancy care but also higher future risk of severe maternal and perinatal morbidity, potentially offering a useful trigger for health system interventions to decrease adverse pregnancy outcomes. US studies have also identified maternal and systemwide factors associated with ED use during pregnancy, including insufficient antenatal care, psychological comorbidity, low socioeconomic status, and substance use, with worse ensuing obstetrical outcomes.9 However, these US studies comprised either commercially insured or low-income patient populations, who may differ considerably from women who receive care within a universally insured health care system, like that in Canada. As another limitation, existing research did not explore prepregnancy ED use, which would, naturally, be exclusive of any condition or concern arising in an index pregnancy. Specifically, studying ED utilization in the 90 days before pregnancy may describe both the health state and health care access of an individual preceding the critical time period of embryonic and placental development. To our knowledge, existing research has also not explored early-pregnancy ED use and its relation to pregnancy outcomes. Finally, prior studies did not measure important maternal or perinatal outcomes that tend to arise after the second trimester, including severe maternal morbidity (SMM), maternal mortality, stillbirth, or severe neonatal morbidity (SNM).Individuals with a greater number of antecedent comorbidities tend to have more peripregnancy ED visits.The current study, undertaken within a Canadian health care system and where all residents receive universally insured care, examined the association between prepregnancy ED use and important adverse maternal and perinatal outcomes. These same outcomes were further assessed in relation to ED use within the first trimester of pregnancy.STROBE) checklist for observational cohort studies.This retrospective population-based cohort was completed within all of Ontario, Canada’s most populous province, with universal health care coverage and standardized collection of all outpatient, ED, and inpatient hospital care services. The use of data in this project was authorized under section 45 of Ontario’s Personal Health Information Protection Act, which does not require review by a Research Ethics Board or informed consent from participants. Reporting of the study findings was consistent with the Strengthening the Reporting of Observational Studies in Epidemiology  number, non-Ontario residents, multifetal births, deliveries before 20 weeks’ or after 42 weeks’ gestation, and liveborn infants discharged alive without a valid OHIP number , the Same Day Surgery Database and National Ambulatory Care Reporting System (NACRS) database, the OHIP claims database, and the ICES MOMBABY database, which links the DAD hospital admission records of delivering mothers and their newborns4The main exposure was any prepregnancy ED encounter within 90 days preceding time zero, which was the estimated conception date of the index pregnancy minus 2 weeks. The reason for setting time zero to 0 weeks’ gestation  was to account for any potential inaccuracy in pregnancy dating and to ensure that the individual was not yet pregnant. An ED is defined as a hospital facility that serves unscheduled patients whose conditions may require immediate care and must be staffed by a physician at all times. An ED visit is defined as an encounter in the ED between a patient seeking care and a physician or other health care professional . A secondary study exposure was any ED visit in the first trimester of pregnancy, between 0 to 12 completed weeks’ gestation, when ED use tends to peak.All ED visits were identified in the NACRS database, defined as an ED encounter between a patient seeking care and a physician or physician assistant or nurse practitioner working under a physician’s supervision  and 95% CIs for the association between a participant having any vs no ED visit within 90 days before pregnancy and each respective study outcome. In the case of more than 1 pregnancy in the same participant during the study period, generalized estimating equations with an exchangeable correlation structure accounted for correlated errors. RRs were adjusted for maternal age, neighborhood income quintile, and neighborhood rurality. An additional analysis further added to the multivariable models the number of comorbidities at the start of the index pregnancy, expressed as the total number of Johns Hopkins ACG System Aggregated Diagnosis Groups . A priori, it was understood that the number of ADGs would likely be collinear with a participant’s likelihood of an ED visit before pregnancy and would markedly attenuate the observed RRs. Post hoc, we also stratified by the number of ADG groups  to assess whether the association between a prepregnancy ED visit and SMM or SNM was consistent across ADG groups.For the main analyses, modified Poisson regression with a robust error varianceNext, in a dose-response analysis, the risk of SMM and SNM were each evaluated in relation to the number of ED encounters in the 90-day prepregnancy period. The number of ED visits was categorized as 0 (reference group), 1, 2, or 3 or more visits, and otherwise modeled as in the main analyses.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Canada (ICD-10-CA).23 Accordingly, the RR for SMM was also assessed in relation to the Main Problem at a participant’s latest  ED visit within the 90-day prepregnancy period, relative to those with no ED visit (reference group). These results were further stratified by gravidity (nulligravid vs gravid) and by parity (nulliparous vs parous) preceding the index pregnancy. The median (IQR) number of days between the last prepregnancy ED visit and time zero were also calculated for each.In NACRS, each ED visit is assigned a “Main Problem” , which is grouped into one of the existing chapters of the P < .05, and all tests were 2-tailed. All cell sizes of 1 to 5 were suppressed to prevent patient re-identification. A sample size calculation was not conducted prior to commencing this study, as all available births in the study period were included.Analyses were conducted using SAS statistical software version 9.4 for Unix (SAS Institute) and the Johns Hopkins ACG System Version 10. Statistical significance was set at There were 2 255 940 livebirths and stillbirths identified, of which 125 695 births (5.6%) were excluded, owing to multifetal delivery, invalid OHIP number, duplicate delivery, extreme maternal age, or non-Ontario residency  had an ED visit within 90 days before pregnancy, occurring at a median (IQR) of 48 days (26-69 days) preceding time zero. In contrast to those who did not visit the ED before pregnancy, women who visited the ED were more likely to be younger, reside in a lower income or rural area, and to have a greater number of ADGs . AntenatWomen who had a prepregnancy ED visit had a higher rate of SMM (22.3 per 1000) than those without an ED visit (16.5 per 1000), equivalent to an unadjusted RR of 1.34 , an adjusted RR (aRR) of 1.37 , and an adjusted risk difference of 6.0  . This riA dose-response association was seen between the number of pre-pregnancy ED visits and the risk of SMM . CompareEach of the main diagnostic groups at the latest prepregnancy ED visit was associated with a higher risk of SMM, with the highest aRRs seen for diseases of the blood ; endocrine, nutritional, and metabolic systems ; and the circulatory system  . Of noteA total of 367 160 women had had an ED visit in the first trimester of pregnancy. This was also associated with a higher risk of SMM, compared with those with no ED encounter during that time  .There were 68 maternal deaths in the entire cohort (3.2 per 100 000 births). Of these, 11 (16.2%) had a prepregnancy ED visit.The risk of SNM was higher among liveborn infants whose mother used the ED within 90 days before pregnancy (68.2 per 1000) than whose mother did not have an ED encounter (55.4 per 1000), for an aRR of 1.24  . The samApproximately 1 in 10 individuals who gave birth in Ontario had an ED visit within 90 days preceding the start of that index pregnancy, while 1 in 6 had an ED visit in the first trimester. Such ED use was associated with a higher risk for severe adverse maternal and perinatal outcomes arising later in pregnancy, especially as the number of ED visits increased.25 An ED visit during pregnancy is more likely among women younger than 25 years, nulliparous women, those residing in a low income or rural area, nonimmigrant women, and those with a greater number of ADGs.4 Rates of SMM in women with and without prepregnancy ED visits differed slightly in this Ontario population cohort than in some US studies,26 partly due to differences in the definition of SMM. For example, some US studies do not include major transfusion within the SMM composite and only evaluate SMM at the time of delivery, as opposed to up to 42 days thereafter.The current findings are consistent with prior research describing a positive association between the presence of maternal comorbidities and other socioeconomic factors and ED utilization.28 but not all,29 studies. Moreover, women with recurrent miscarriage and infertility problems—often associated with polycystic ovarian syndrome and endometriosis—have a higher prevalence of chronic conditions, such as obesity, diabetes, and hypertension.30 Although less prevalent, prepregnancy ED visits for a hematological, endocrine, or circulatory condition was associated with the highest RR of SMM  for preterm birth.25 In the current study, prepregnancy ED use showed a higher risk of SNM among both nulliparous and parous mothers, and we observed a dose-response association.The current study introduces novel information about prepregnancy ED use and adverse perinatal outcomes. With ascertainment of the study exposure prior to the onset of pregnancy, the possibility of reverse causation was very low. In a retrospective cohort study of 107 207 Medicaid recipients in North Carolina who had a livebirth, 58% sought ED care 1 or more times during pregnancy, which was associated with a higher rate of preterm birth.24 Yet, even after adjusting for maternal age, neighborhood income and rurality, prepregnancy and first-trimester ED use remained associated with SMM and SNM. This is underscored by the fact that, across all diagnostic categories, ED utilization was associated with worse outcomes for mother and fetus, and especially, with a higher number of antecedent ED visits. Post hoc, higher rates of SMM and SNM were seen as the number of ADG-denoted comorbidities before pregnancy increased, even though the aRRs were similar  in perinatal mortality from group-based interventions that link prepregnancy with pregnancy care.37Efforts to improve preconception care should elaborate why so many women of reproductive age access the ED and what their needs are. Among women with chronic noncommunicable conditions, existing studies suggest a proactive approach of routinely asking those women about their pregnancy intentions and providing both advice on how to avoid unplanned pregnancy and specific recommendations about how to optimize their health once they wish to conceive. These women also want advice from peers with a similar health condition, including through online and social media.38 Even so, as preconception care applies not only to those planning a pregnancy, but also those who could become pregnant, a prepregnancy ED visit may be an opportunity for positive intervention. One example is oral iron supplementation among those with anemia.39 For those with prepregnancy diabetes, a preconception care bundle is associated with improved maternal and perinatal outcomes.40 And in those with chronic hypertension in pregnancy, blood pressure control (<140 mm Hg systolic blood pressure and <90 mm Hg diastolic blood pressure) is associated with fewer serious maternal complications.41Initiatives are also needed that enable an individual of reproductive age or who has a potentially viable pregnancy to receive timely care within a primary care and/or specialty clinic, rather than the ED.4This study has limitations. It only included pregnancies ending in hospital livebirth or stillbirth, so the association between ED use and miscarriage or induced abortion was not assessed. ED use in pregnancy and the postpartum period has previously been observed to occur among 36% of livebirths, 47% of stillbirths, 74% of miscarriages, and 85% of threatened abortions.16 We did not have information about race or ethnicity, which were not controlled for, and which have been shown to influence pregnancy-related ED utilization.7 Prior US studies correlated low socioeconomic status and substance use with worse pregnancy outcomes,9 but we could not account for smoking or other substance use.42 While the current study preceded the SARS-CoV-2 pandemic, fluctuations in ED use that may have been seen during the pandemic appear to be returning to prepandemic levels.44Details were lacking about the severity of participants’ conditions at their ED presentation or the pattern of care they received within the ED or thereafter. Whether a pregnancy was planned was not known, and deliveries occurring outside of a hospital setting were not captured, such as midwifery homebirths, which comprise fewer than 1% of livebirths in Ontario.15 The fact that this study included all women in Ontario afforded care under a single-payer health care system likely reduced participant selection bias and information bias. Despite adjusting for certain maternal factors, and further adjusting for antecedent comorbidities, the potential remains for residual confounding between prepregnancy ED use and adverse maternal and perinatal outcomes, as discussed previously in the Discussion section.By setting time zero at 2 weeks before conception, we ensured that each prepregnancy ED encounter was exclusive of the index pregnancy. As the latest ED visit occurred 48 days  before the start of the pregnancy, this was likely true. Pregnancy dating in Ontario is likely to be accurate for most women who give birth.In this study, ED utilization was common both in the prepregnancy period and in early pregnancy. ED use may not only reflect an individual’s access to prepregnancy care but may offer a pragmatic early alert about an individual’s higher risk of SMM and SNM. Future studies could assess whether ED use is a useful trigger for health system interventions designed to decrease adverse pregnancy outcomes.