OPRM1 gene and the risk of increased VAS scores in patients with colorectal cancer who underwent laparoscopic radical resection for which fentanyl was used.This study was conducted to examine the association between the A118G polymorphism of the OPRM1 A118G genotype in subjects were detected. The relationship between the A118G polymorphism of the OPRM1 gene and increased Visual Analogue Scale (VAS) scores throughout the perioperative period was explored. A total of 101 patients receiving fentanyl anesthesia undergoing laparoscopic radical resection of colon tumors at Zhongshan Hospital, Fudan University between July 2018 and December 2020 were investigated in the present study. The relative risk between the A118G polymorphism of the OPRM1 gene and VAS ≥ 4 in the PACU was estimated using the adjusted effect relationship diagram, baseline characteristic analysis, and multiple logistic regression analysis. The relationship between the A118G polymorphism of the OPRM1 gene and VAS in the PACU, as well as perioperative fentanyl usage, was examined after confounders were adjusted.The OPRM1 A118G wild gene A were less sensitive to fentanyl, which was a risk factor for PACU VAS ≥ 4. Before the model was adjusted, the odds ratio (OR) was 14.73 (P = 0.001). After adjusting for age, sex, weight, height, and the duration of surgery, the OR increased to 16.55 (P = 0.001). When adjusting for age, sex, weight, height, surgery duration, COMT Val158Met gene polymorphism, CYP3A4 *1G gene polymorphism, and CYP3A5 *3gene polymorphism, the OR was 19.94 (P = 0.002). Moreover, OPRM1 A118G wild type gene A was found to be a risk factor for increased dosage of fentanyl in the PACU. Before the model was adjusted, the OR reached 16.90 (P = 0.0132). After adjusting for age, sex, body weight, intraoperative fentanyl dosage, surgery duration, and height, the OR was 13.81, (P = 0.0438). When adjusting for age, sex, weight, height, intraoperative fentanyl dosage, surgery duration, COMT Val158Met gene polymorphism, CYP3A4 *1G gene polymorphism, and CYP3A5 *3 gene polymorphism, the OR reached 15.23, (P = 0.0205).Subjects with OPRM1 gene carrying wild gene A was a risk factor for VAS ≥ 4 in the PACU. Moreover, it is a risk factor for increased dosage of fentanyl in the PACU.The A118G polymorphism of the The online version contains supplementary material available at 10.1186/s12871-023-02120-1. Effective pain management following surgery is critical to accelerating postoperative recovery, shortening the hospitalization period, and improving patients’ satisfaction . At presOPRM1). One of its SNPs, named A118G, is prevalent and crucial in OPRM1, which results in an amino acid change in the extracellular glycosylation site of MOR cells.OPRM1 A118G polymorphism can affect the function of MOR [CYP3A4*1G, COMT Val158Met and CYP3A5 *3 are closely related to the analgesic effect of opioids.Many studies have shown that individual differences in fentanyl response are closely related to human genetics. Mu-opioid receptor (MOR) is the main target of fentanyl and is encoded by the human opioid receptor-1 gene  class I-II.Exclusion criteria: intraoperative switch from a laparoscopic surgery to laparotomy; intracorporeal anastomosis following laparoscopic surgery; patients with morbid obesity or extreme emaciation (BMI > 30 or < 20 kg/m2), liver and kidney dysfunction, and chronic pain; patients who have taken enzyme inducers and inhibitors that affect CYP3A4 enzyme activity within 1 month.Withdrawal criteria: subjects asked to withdraw informed consent, change in operation mode during operation, the intraoperative blood loss reaching more than 500 ml.Data were obtained from patient medical records. The following variables were considered in our modeling: age, weight, height, intraoperative fentanyl dose, operation time, gender, and genotype. Other variables, such as comorbidity/ASA, and tumor location (right/left) were supplied in Supplementary Material 1 .Patient-controlled intravenous analgesia (PCIA) was performed with the PCA pump. The pump was attached to the intravenous tube after the tracheal tube was removed. The patient was observed in PACU for one hour. If the visual analogue scale/score (VAS) ≥ 4, the patient-controlled dose of fentanyl, i.e. 20 μg, was delivered. If necessary, it could be given repeatedly until the VAS was less than 4. The analgesic effect and adverse reactions were observed in PACU.Blood pressure, heart rate, pulse, oxygen saturation, Electrocardiogram, and other vital indicators of the patients were all monitored. The following domains were recorded: the fentanyl consumption; and the VAS from the beginning of surgery to 48 h after surgery.The primary endpoint was that the spontaneous breathing rate of patients was stable at ≤ 10 times/minute before the removal of the tracheal tube, and the secondary endpoint was VAS of less than 4 when the patient left PACU.Main instruments and reagents included Applied Biosystems 7500 Fast Realtime PCR System (Thermo Fisher Scientific (China) Co., Ltd.), AB3730 sequencer , Lab-Aid 820 Midi reagent for nucleic acid extraction , Permix Taq@Hot Start Version  Co., Ltd.), and sequencing reagent (Thermo Fisher Scientific (China) Co., Ltd.).Two milliliters of EDTA-K2 anticoagulant were collected before operation. Lab-Aid 820 Midi reagent was used for DNA extraction in strict accordance with the instructions. The DNA was then stored at -20℃ until detected. The software Oligo7 was used for PCR primer design, as shown in Table 1Reaction conditions: 94 ℃ for 5 min; 45 cycles ; 72 ℃ for 5 min; insulation at 4 ℃. After purification, the PCR amplification products were sequenced on ABI 3730 DNA Analyzer, and the results were analyzed by the Chromas software.The present study conformed to the principles of the Declaration of Helsinki. Approval was obtained from the Research Ethics Committee of the Zhongshan Hospital, Fudan University .OPRM1, CYP3A4, COMT, and CYP3A5 polymorphism. R version 3.4.1 and the software IBM SPSS (version 21.0) were used to perform statistical analysis. All P values for statistics were 2-tailed, and P < 0.05 was regarded as statistically significant.Data are expressed as mean (SD) for continuous variables and percentage (%) for categorical variables. The continuous variables included age, weight, height, intraoperative fentanyl dose, and operation time. The categorical variables included gender, and genotype. The dose–response curve was plotted with the curve-fitting method. The multivariable regression model included additional variables such as age, weight, height, the intraoperative dose of fentanyl, operation time, gender, and OPRM1 A118G, CYP3A4 *1G, CYP3A5 *3, and COMT Val158Met.Figure CYP3A4 *1G, CYP3A5 *3 or COMT Val158Met between VAS < 4 and VAS ≥ 4, as shown in Table There was no significant difference in age (59.53 ± 5.87 vs 61.67 ± 4.00), gender, weight (62.23 ± 9.17 vs 64.22 ± 7.60 kg), height (164.80 ± 7.77 vs 162.89 ± 6.53 cm), fentanyl dosage (8.95 ± 2.02 vs 10.21 ± 2.28 μg/kg), operation time (2.33 ± 0.54 vs 2.26 ± 0.33 h), OPRM1 genotypes between the two groups. Patients carrying wild gene A had decreased fentanyl sensitivity and were more prone to VAS ≥ 4.Among 101 subjects, the number of VAS ≥ 4 and VAS < 4 was significantly different. There was significant difference in OPRM1 A118G、CYP3A4 *1G、CYP3A5 *3 and COMT Val158Met polymorphisms, as shown in Table The feature selection factors with significant differences in univariate analysis were included in the regression analysis. Based on univariate analysis, multivariate logistic analysis was performed for each factor, including age, sex, weight, height, intraoperative fentanyl dosage, operation duration, OPRM1 A118G polymorphism, and was a risk factor for VAS ≥ 4. Further regression analysis showed that without adjusting of confounders, the VAS ≥ 4 in GA and AA-type subjects was 13.73 times higher than that of GG-type subjects. After fully adjusting the confounding factors that may affect PACU VAS, such as age, sex, weight, height, and operation duration, carrying gene A of OPRM1 is a risk factor in VAS ≥ 4. Subjects carrying gene A of OPRM1 were 15.55 times higher than GG-type subjects. After adjusting of age, sex, weight, height, operation duration, and the polymorphisms of CYP3A4*1G, CYP3A5*3, and COMT Val158Met, the probability of VAS ≥ 4 in carrying gene A of OPRM1 subjects was 18.94 times higher than that in GG type subjects, as shown in Table Carrying wild gene A was associated with VAS ≥ 4, according to the CYP3A4 *1G、CYP3A5 *3 and COMT Val158Met, the probability of PACU VAS ≥ 4 in patients carrying gene A of OPRM1 is significantly increased.Figure CYP3A4 *1G, CYP3A5 *3 and COMT Val158Met polymorphisms, the risk of PACU VAS ≥ 4 in the subjects carrying gene A of OPRM1 A118G is significantly increased. In the GG group, the adjusted mean was 0.036,(95%CI: 0.002–0.366); in the GA + AA group, the adjusted mean was 0.468,(95%CI: 0.096–0.879).Adjusted according to age, sex, weight, height, intraoperative fentanyl dosage, operation duration and the polymorphisms of OPRM1 A118G was associated with fentanyl dosage during PACU. Carrying A gene is a risk factor for decreased fentanyl sensitivity and increased fentanyl dosage during PACU. Further regression analysis showed that the probability of GA and AA-type subjects receiving high-dose fentanyl was 15.90 times higher than that of GG-type subjects; After adjusting for age, sex, weight, height, intraoperative fentanyl dosage and operation duration, the probability of GA and AA-type subjects was 12.81 times higher than that of GG-type; After fully adjusting for age, sex, weight, height, intraoperative fentanyl dosage, operation duration, and the polymorphisms of CYP3A4 *1G, CYP3A5 *3, COMT Val158Met, the probability of subjects carrying A gene was 14.23 folds higher than that of GG homozygous, as shown in Table The polymorphism of CYP3A4 *1G, CYP3A5 *3, and COMT Val158Met, the dose of fentanyl in patients carrying gene A of OPRM1 is significantly increased in PACU. In the GG group, the adjusted mean was 43.10 μg(95%CI: 33.82–52.37); in the GA + AA group, the adjusted mean was 59.01 μg(95%CI: 45.36–72.65).The adjusted effect relationship diagram in Fig. With the advancement of the gene and the in-depth investigation of anesthetic medications, more and more studies have revealed that gene polymorphism would result in varying effects of the same anesthetic agent on different populations and individuals . Gene poFentanyl is a potent synthetic analgesic with an analgesic mechanism similar to morphine . FentanyOPRM1 genotype differed significantly between populations with VAS ≥ 4 and VAS < 4. The subjects carrying gene A are less sensitive to fentanyl and more prone to VAS ≥ 4.The VAS scale was employed in this study to assess the pain level of patients during operation . VAS is OPRM1). OPRM1 A118G mutation is the most common and reported functional gene. Some studies have shown that its gene mutation reduces the efficacy of various opioids, and patients with GG need more opioids to relieve pain [OPRM1 A118G mutation can enhance the analgesic effect of patients [The analgesic efficacy and adverse reactions of opioids mainly act on three opioid receptors known as δ, κ, and μ. Among them, μ opioid receptors are the key targets of endogenous and exogenous opioid analgesia, tolerance, dependence and other effects, which are coded by the μ Receptor gene  of our study may affect the decision about which and how many factors were included in the adjusted analysis. And the stability and applicability of the adjusted model needs to be verified and evaluated in future cohort studies with larger sample sizes.OPRM1 was highly associated with an increase in fentanyl dose and VAS ≥ 4 in the PACU. It is important for the clinical to pay attention to gene detection in anesthetic drugs before the operation. It has provided a novel way to analyze the influencing factors of VAS, and may be the valuable clinical significance for selecting a suitable anesthetic scheme.Our findings revealed that carrying the A gene of Additional file 1.


Silk fibroin is a natural polymer with physicochemical properties heavily dependent on its silkworm sources and cultivation conditions. Hence, this study critically compared the characteristics and capacity to generate micro-/nanoparticles of fibroin extracted from the Thai silk and Vietnamese silk. Both Thai fibroin (SFT) and Vietnamese fibroin (SFV) were extracted and fabricated into micro-/nanoparticles using the same methods of desalination and condensation, respectively. Firstly, the amino acid compositions of SFT and SFV were determined and found to be similar, suggesting that the different cultivation conditions did not alter the fibroin chemical contents. Secondly, utilizing various analytical techniques, the SFT structure revealed less heavy chains, more light chains and P-25 glycoproteins, and lower crystallinity than those of SFV. Accordingly, compared to the particles formed by SFT, the SFV-based particles were significantly bigger (∼1700 nm vs. ∼150 nm), and possessed less drug (Amphotericin B) entrapment efficiency (64.3 ± 4.4% vs. 79.3 ± 5.1%), higher hemototoxicity, and less biostability in the blood. Conclusively, these differences add more insights for the appropriate applications of each fibroin kind to best promote its qualities and effectiveness.  Silk fiears ago . Generalears ago . The heaears ago ,7. For lears ago . Althougears ago . These dears ago ,8.Fibroin, due to its natural biodegradability, biocompatibility, and structure self-modifiability, has been utilized in various drug delivery systems namely hydrogels, films, microparticles, and nanoparticles, for the administration by oral, parenteral, transdermal, and ophthalmic routes ,10,13,14Bombyx mori L. domesticated silkworm species, like other animals, generate varying silk quality under different growth circumstances, which influences fibroin characteristics. The nutrition of mulberry leaves , the main feed of Bombyx mori L., and the living environment such as temperature, humidity, ventilation, and light, have significant impacts on the growth and development of silkworm larvae and cocoons [The  cocoons . Typical cocoons ,16. Firs cocoons . Secondl cocoons ,18. Last cocoons . In ThaiTaken these aforementioned issues into consideration, to assess the impact of raw materials on the drug delivery system properties, this study, for the first time, critically investigated the differences, focused on the fibroin crystallinity and related properties, between the Thai and Vietnamese silks. Representative products made from these fibroins, micro-/nanoparticles employing amphotericin B (AmB) as a model drug, were also compared. AmB was selected because (1) it is a water-insoluble lipophilic drug that commonly requires a carrier such as nanoparticles to effectively deliver it to the sites of actions , and (2)22.1Bombyx mori L. silkworm cocoons were employed in this investigation. The Thai dried silkworm cocoons were supplied by Bodin Thai Silk Khorat Co., Ltd, Nakhon Ratchasima, Thailand, and the Vietnamese dried silkworm cocoons were obtained directly from farmers in Truc Ninh, Nam Dinh, Vietnam. Both types of cocoons were selected at the same growth phase (40 days after the eggs hatched), with similar appearances, shapes, and physical properties. Silkworm cocoons were gathered and maintained at room temperature after silkworm pupae were removed. All other storage and delivery conditions were kept comparable between the two silk types. The sheep blood was bought from Nam Khoa Limited Liability Company, Vietnam , with ISO 9001, ISO 13485, and WHO GMP standards, and completely used within 01 week. AmB was imported from Sigma-Aldrich, Singapore. The chemicals sodium carbonate (Na2CO3), calcium chloride (CaCl2), calcium nitrate (Ca(NO3)2), and ethanol 99% were supplied by Xilong, China. The dialysis tubing cellulose membrane (12000 MWCO) was provided by Sigma-Aldrich, Singapore. Other chemicals were of reagent grade or higher.Thai and Vietnamese The utilized instruments were UV–Vis spectrophotometer , benchtop centrifuge , freeze-dryer , magnetic stirrers , infrared spectrometer , X-ray diffractometer , differential scanning calorimetry machine , zeta potential analyzer , and scanning electron microscopy .2.22CO3 0.5% solution at 100 °C for 1 h, followed by washing thrice with deionized (DI) water and drying at ambient temperature. Then, the degummed silk was dissolved in mixture of CaCl2:H2O:Ca(NO3)2:EtOH with weight ratio of 30:45:5:20 at 90 °C to obtain the fibroin solution. Finally, the fibroin solution was dialyzed at room temperature for 3 days, and the product was lyophilized at −40 °C. The silk fibroin was preserved at 4 °C until further uses (The fibroin was extracted from the Thai (SFT) and Vietnamese (SFV) silk cocoons using the protocol developed by our group ,21. To ther uses .Fig. 1Si2.3The FNP from SFT and SFV were prepared by the simple desolvation method . A total2.4The characteristics of the SFT and SFV were determined and compared, in terms of physical properties, amino acid compositions, molecular weights, molecular structures, and crystallinity.2.4.1The physical properties, in terms of appearance, texture, and stability, of the SFT and SFV were observed and reported as images.2.4.22HPO4 and 10 mM Na2B4O7 (pH 8.2) (B) with a gradient running mode . The process was fully validated followed the International Conference on Harmonisation (ICH) guideline.The amino acid compositions of SFT and SFV were determined utilizing the high performance liquid chromatography  with an automatic sampler, a reverse-phase column XDB C18 (250 mm × 4.6 mm x 5 μm), a fluorescence detector (excitation wavelength of 260 nm and emission wavelength of 320 nm), an injection volume of 10 μL, a flow rate of 1.75 mL/min, and a mobile phase consisted of acetonitrile (A) and 10 mM NaTo obtain the test samples, the fibroin freeze-dried powder (0.5 g) was hydrolyzed by 10 mL of 6 N HCl for 24 h in the nitrogenic atmosphere at 110 ± 5 °C, followed by pH adjustment to 1–3 by 50% w/v KOH. Then, the resulted amino acid solutions were reacted with Fmoc N-hydroxysuccinimide ester at 40 °C for 30 min to form the fluorescent derivatives. Finally, the samples were subjected to HPLC analysis and the percentages of each amino acid in fibroin molecules were calculated based on standard of the respective reference amino acids.2.4.3The molecular weights of the SFT and SFV were assessed by protein sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, following standardized protocol with minor modification . Briefly2.4.4The molecular and chemical structures of SFT and SFV were measured using standard analytical methods of Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC).2.4.4.1−1) with KBr pellet method. The spectra, collected at 4.0 cm−1 resolution, were measured in a desiccated air purge at coaddition of 256 interferograms. The empty KBr pellet was used to determine background signals and subtracted from the sample reading.The FTIR analysis of the SFV and SFT was performed on a NICOLET 6700 (Thermo) spectrophotometer (wavenumber ranging from 4000 to 500 cm2.4.4.2The XRD was measured on a D8 Advance XRD instrument (Bruker), using Cu Kα radiation generated at 45 kV and 36 mA (λ = 0.154 nm). The lyophilized SFV and SFT were spread out onto quartz substrates and analyzed from 5 to 50° (2θ) at scan speed of 2°/min.2.4.4.3The DSC analysis was conducted using DSC 200 F3  equipped with a ceramic:FRS6 sensor, and proceeded at a temperature range of 0–300 °C and a scanning rate of 3 K/min, in nitrogen gas flow of 20 mL/min. The lyophilized SFV and SFT (5–8 mg) was subjected in an aluminum pan, and measured with a blank pan as reference.2.4.5IRI, D1622, and D1646 are the CI values, the crystalline portions density, and the amorphous portions density of the amide I bands. Similarly, CIIRII, D1517, and D1560 are the CI values, the crystalline portions density, and the amorphous portions density of the amide II bands.The fibroin crystallinity was assessed by a value called crystallinity index (CI), defined as the crystalline fractions/ratios of a material/formulation and calculated by dividing the crystalline signal intensities by the sum of the crystalline and amorphous signals' intensities . In this2.5The micro-/nanoparticles made from SFT and SFV were characterized and compared their properties in terms of size and zeta potential, morphology, structure and crystallinity, drug entrapment and release profile, biostability, and hematotoxicity.2.5.1The particles sizes and polydispersity indexes (PI) were determined by dynamic light scattering (DLS) method, whereas the zeta potentials were measured by phase analysis light scattering (PALS) method. The instrument was operated at a fixed wavelength of 632.8 nm with a BI-200SM Goniometer linked with a BI-9010AT digital correlator. The zeta potential values were calculated using the Smoluchowski equation.2.5.2The particle morphology was observed using field emission scanning electron microscopy . The FNP-SFT and FNP-SFV lyophilized powders were re-dispersed in DI water, and the dispersions were dropped on a SEM stub with aluminum tape, followed by drying at ambient temperature, gold-coated, and subjected to FESEM analysis. The images were obtained using the software included in the machine.2.5.3The structure and crystallinity of FNP-SFT and FNP-SFV were determined and calculated using the FTIR technique, in similar approach and experimentation with the respective fibroin properties (section 2.4.4 and 2.4.5).2.5.42 = 0.9990), and the AmB entrapment efficiency was calculated using equation To determine the drug entrapment efficiency of the FNP-SFT and FNP-SFV, the indirect method was employed. To this end, the remaining AmB, after being loaded into the particles, was separated by centrifugation, and UV–Vis spectroscopic measured at 408 nm using a Jasco V730 spectrophotometer. The unloaded AmB amount was then determined based on AmB standard curve , V is the withdrawal sample volume at each time point (1 mL), M0 is the initial amount of AmB, and Mi is the total amount of withdrawal AmB at the time point i.The shake-flask technique was used to determine the release characteristics of AmB loaded FNP-SFT and FNP-SFV. The phosphate buffer pH 6.8 containing 10% w/v Tween 80 was used to establish the sink condition  . The lyo2.5.6The biostability of FNP-SFT and FNP-SFV in the biological blood was determined using the weight-loss experiments . Briefly2.5.7The hematotoxicity, determined by the hemolysis percentage, of the FNP-SFT and FNP-SFV was in-vitro investigated on the sheep red blood cells ,8. To th2.6All experiments, both qualitative and quantitative, were conducted in at least three times. The comparisons between samples, if any, were performed by the analysis of variance (ANOVA) tests, with p values of less than 0.05 for significant associations.33.13.1.1The fibroin amounts extracted from the two varieties of silk cocoons were significantly different. Utilizing the same extraction procedure, the extraction efficiencies for Thai silk and Vietnamese silk were 58.65 ± 3.12% and 20.08 ± 2.47%, respectively. Since different silk types with distinct silkworm living environments and cultivation conditions yield significantly different fibroin and sericin content , our res3.1.2The amino acid compositions of SFT and SFV are presented in 3.1.32CO3, the salt alkaline nature might lower the macromolecules weights in the peptide chain, thus influencing the weight of the produced fibroin [Generally, the fibroin molecule composes of a heavy chain ∼390 kDa) and a light chain (∼26 kDa) joined by a disulfide bridge, and non-covalently bound by a P-25 glycoprotein 90 kDa an,30. For  fibroin . In this3.1.4−1, 1515 cm−1, and 1241 cm−1, respectively. These peak locations have been confirmed to be the crystalline portions of the fibroin water-insoluble silk-II polymorph [To elucidate the molecular structures of SFT and SFV, analytical methods of FTIR, XRD, and DSC were employed. Firstly, the FTIR spectra of both fibroin demonstrated characteristic fibroin peaks consisting of five main amide bands  A. The coolymorph . Therefoolymorph , while tolymorph . Interesolymorph ,31. Thisolymorph . ConclusSince fibroin is a semi-crystalline material composed of both crystalline and amorphous regions, its crystallinity could be quantitatively calculated based on the FTIR spectra . To thisIn summary, taking all data into consideration, it could be concluded that both SFT and SFV possessed similar amino acid compositions, indicating that the geographical and cultivation conditions did not affect much on the fibroin chemical contents. On the other hand, the fibroin structures were significantly different between the two silk types. The SFV structures contained both heavy and light chains, and P-25 proteins, thus mainly possessing a crystalline silk-II polymorph. Conversely, the SFT structure mostly composed of light chains and P-25 proteins, thus mainly having the amorphous silk-I polymorph. In the next section, the FNP formulated from these two distinct fibroin were thoroughly investigated and compared.3.23.2.1Utilizing the same preparation method, the FNP were fabricated from SFT and SFV, and characterized and compared their properties. In terms of the particle sizes, the DLS analysis revealed that the particles produced from SFT are, however, many orders of magnitude smaller  than those prepared from SFV, which possessed a micro-ranged sizes of 1200–2500 nm A and C. 3.2.23.2.3The ability to encapsulate and release the loaded drugs of FNP-SFT and FNP-SFV were further investigated, using the model drug AmB. To this end, the FNP-SFT and FNP-SFV possessed a drug EE% of 79.3 ± 5.1% and 64.3 ± 4.4%, respectively. This could be due to the high crystallinity of the FNP-SFV particles, with high β-sheet structure, consequently restricting the drug's entry into the particles and lowering the loading efficiency. Nevertheless, our results were different than those of the previous reports, which indicated that FNP with higher crystallinity showed better EE% than that of the particles with lower crystallinity, using the model drug of alpha mangostin and paclitaxel . TherefoRegarding the AmB release profiles from the particles. Although the experiments were performed in the sink condition, in which the AmB solubility was not a factor affecting its release rate, no significant AmB amount was released from both particles (<4%). Our results were in agreement with the previous report that encapsulating the AmB in the FNP . This mi3.2.4The fibroin structure, as well as its crystallinity, have been proven to significantly affect the FNP bio-properties ,8. Thus,Regarding the biostability, the FNP-SFV degraded three times faster than FNP-SFT in the first 3 h, and this rate steadily dropped with time. The quick breakdown of FNP-SFV particles could be explained by their large particle size and porous shape, making the exterior bonds become weaker, thus allowing the blood proteolytic enzymes to easily come into contact and break down the fibroin molecules.4The study discovered significant variations in the characteristics of Thai silk fibroin and Vietnamese silk fibroin, as well as their corresponding fibroin micro-/nanoparticles. The main differences between these two types of silk fibroin, and micro-/nanoparticles, are summarized in Ngoc Yen Nguyen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Thi Ngoc Phuong Nguyen: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Nguyen Ngoc Huyen, Van De Tran & Tran Thi Bich Quyen: Performed the experiments; Wrote the paper.Huynh Vu Thanh Luong: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Duy Toan Pham: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.Data will be made available on request.10.13039/501100005645Ministry of Education and Training under grant number B2022-TCT-04.This research is funded by the Vietnam The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Furniko flour (FF) is a traditional roasted flour derived from a maize landrace, commonly consumed by Greeks of Pontic origin in the northern regions of Greece. Despite its perceived nutritional benefits, there is a lack of scientific data to support and highlight its value. This study aimed to compare the nutritional, physicochemical, anti-nutritional, functional, and antioxidant characteristics of FF with those of traditional and non-traditional types of maize flour. Furniko flour (FF) presented the highest values for protein (10.86 ± 0.36 g/100 g), fat (5.05 ± 0.08 g/100 g), K (539.93 mg/100 g), Mg (126.38 mg/100 g), P (296.4 mg/100 g), Zn (2.44 mg/100 g), and total phenolic content (TPC) (156 mg GAE/100 g). However, FF exhibited lower levels of Fe (3.83 mg/100 g), carbohydrates (70.55 ± 0.24 g/100 g), and antioxidant activity (0.27 ± 0.02 µmol of TE/g) than other types of flour examined. Furniko’s functional properties make it an excellent source for porridges, while its low content of antinutrients reduces the possibility of low bioavailability of Fe, Zn, Mg, and Ca. Due to its significant and functional characteristics, Furniko flour could be considered an important material in the food industry, especially in bakery goods and health-oriented foods like energy bars, breakfast cereals, and gluten-free pasta. More research is needed, however, to thoroughly investigate its dietary potential and compatibility with other components.The online version contains supplementary material available at 10.1007/s11130-023-01078-2. Furniko flour is a popular traditional maize-based food product in Pontic Greek cuisine [ cuisine . The Pon cuisine . Maize,  cuisine , was als cuisine . However cuisine . Interes cuisine .furniko-making process in Supplementary Materials 2. The distinctive flavor of furniko makes it a key ingredient in a dish called chavitz, havits, or havitz  [gofio [For instance, the traditional preparation of furniko flour involves roasting maize on the cob in a wood-fired oven, a process that lasts 24 h and is carried out until the oven’s inner dome turns white, signaling optimal temperature. Keramaris et al.  describeke meal) . Roasted) [gofio .furniko flour. Hence, this study is focused mainly on the quality characteristics of roasted flour prepared by the Pontic Greeks’ traditional preparation method, which involves roasting maize on the cob in a wood-fired oven—a procedure that differs significantly from the laboratory roasting or pan roasting used in earlier studies. The method of preparation can have a significant impact on a food product’s nutritional profile, and it was of great interest to us to research how this unique method affects the nutritional content of the maize flour. Therefore, this study aims to evaluate the nutritional properties of furniko flour and compare them with those of similar flour products.To the best of our knowledge, limited information is available on the nutritional, physicochemical, functional, antinutritional, and antioxidant properties of The section on materials and methods is accessible in Supplementary Materials 1.Furniko flour (FF), non-traditional roasted flour (NTRF), homemade flour (HF), and commercial flour (CF), as listed in Table p < 0.05) amount, likely due to evaporation resulting from heat [Furniko flour (FF) exhibited the highest protein content among all tested samples, a statistically significant difference (p < 0.05). In contrast to Oboh et al. [p < 0.05). Furniko flour (FF) exhibited the highest crude fat content (5.05 ± 0.08 g/100 g), which was significantly higher than that of NTRF at 4.11 ± 0.05 g/100 g and CF at 0.87 ± 0.10 g/100 g. Therefore, roasting seems to increase the fat content of FF, which is in accordance with previous reports [p < 0.05) increase. The ash content in each flour sample indicates the mineral content [Furniko Flour (FF) exhibited the maximum crude fiber content (3.78 g/100 g), supporting previous reports that roasting increases fiber content [p < 0.05) higher content (80.40 ± 0.29 g/100 g) than FF (70.55 ± 0.24 g/100 g). The lower carbohydrate content of the FF, which supports previous research [p < 0.05) the highest energetic value  among all flour samples. Although the energetic value of the traditional FF was 371.03 ± 0.81 kcal/100 g, it was slightly lower despite its elevated protein content. This could be due to its reduced carbohydrate content.The maize flour samples analyzed in this study include traditional rom heat . High-terom heat . The crurom heat , rangingh et al. , 14, posh et al. . A signi reports . The roa reports . The ash reports , with ro content . Throughresearch , can likresearch . The eneThe current study examined nine minerals, namely Ca, K, Mg, Na, P, Fe, Mn, Cu, and Zn. The results are presented in Table per serving [Furniko flour (FF) and HF are better suited for adults, whereas non-traditional roasted flour is an ideal option for children. As per European Food Safety Authority (EFSA) recommendations, FF observed the most significant dietary contribution to phosphorus (P) for both children (47.42%) and adults (43.11%), and to protein (36.2%) for children. The two other flour varieties, NTRF and HF, had higher contributions of phosphorus and iron. It is critical to acknowledge that the nutritional value of the flour samples exhibited variability based on age and flour-making procedures. Overall, the nutritional contribution of FF aligns closely with that of NTRF and HF, with minor variations. All calculated contributions are shown in Tables S3 and S4, presented in Supplementary Materials 2.This study evaluated the nutritional value of the maize flour varieties, demonstrating their contribution to the daily nutrient requirements of both children and adults. Nutrient contributions, which are classified as high 20% or more) or low (less than 5%) % or moreTable S5  shows the fatty acid profile of the four flour samples, including the total saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA), plus UFA/SFA and omega-6/omega-3 ratios. The fatty acid composition was comparable across samples, all of which contained SFA, MUFA, and PUFA. Both CF and FF had palmitic acid as the most abundant SFA, contributing 16.36 and 16.28% of their total SFAs, respectively. Stearic acid was present in modest concentrations (1.45–2.71%) in all samples. Oleic acid was the most abundant MUFA across all samples, accounting for 26.84–34.32% of total MUFAs, followed by eicosapentaenoic and palmitoleic acids, an observation that is consistent with those of earlier studies . The appp < 0.05) in phytic acid content was observed when comparing FF (0.12%) with HF (0.16%). The total oxalate content in the analyzed flour samples varied significantly (p < 0.05), from 0.29% in CF to 2.61% in FF. Although roasting typically reduces oxalate content [Figure S4  illustrates the antinutritional components related to each flour sample. The phytic acid content in the maize flour samples was notably low, ranging from 0.05 to 0.16%. A statistically significant decrease , yet both were significantly (p < 0.05) higher than the other flour samples. The total phenolic content (TPC) in FF was found to be greater than in HF. This is due to the liberation of bound phenols and improved extractability during the roasting process [Table S6, presented in Supplementary Materials 2, highlights the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of the flour samples. The TPC values of the flour samples ranged from 65 mg GAE/100 g for CF to 156 mg GAE/100 g for FF. The TPC values of FF and HF were not significantly different , while unroasted flour samples HF and CF both exhibited lower values (0.19 µmol of TE/g). Antioxidant activity, via the FRAP assay, revealed that NTRF presented significantly (p < 0.05) the highest activity (11.56 µmol TE/g), while CF presented the lowest activity (1.85 µmol TE/g). Roasted flour samples showed higher antioxidant activity compared to their unroasted counterparts, supporting the results reported by Oboh et al. [r = 0.123, not significant) and FRAP , and a strong positive correlation between DPPH and FRAP . These results suggest that there is a potential link between total phenolic content and antioxidant activity. Moreover, DPPH and FRAP are closely related. On the other hand, TFC exhibited a negative correlation with FRAP . This strong negative correlation suggests a possible inverse relationship between the flavonoid content and the antioxidant capacity as determined by FRAP. Additionally, TFC had a weak negative correlation with DPPH . Finally, TPC and TFC had a moderately negative correlation . These results highlight the nuanced interaction between phytochemical content and antioxidant activity in samples of maize flour.The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays were utilized to assess the antioxidant activity of the flour extracts. NTRF and FF presented the highest levels of radical scavenging activity among all flour types, whereas HF and CF showed the lowest levels by the application of the DPPH assay. As y p < 0.0 the highh et al. . These rh et al. . Pearsonp < 0.05) observed in NTRF. Unexpectedly, the water activity of FF was significantly (p < 0.05) higher than that of HF. This may be attributed to improper flour packaging that allowed the absorbance of ambient moisture. In terms of pH, the roasted flour varieties showed significantly (p < 0.05) lower values  compared to the unroasted flour samples . These pH ranges obtained from the studied flour samples are consistent with data reported for blue [Food storage properties are improved by thermal processing, such as extrusion and roasting . In thisfor blue  and yellfor blue  maize.p < 0.05) difference observed among the roasted and unroasted flour samples. Commercial flour (CF) had the lowest WAC, whereas NTRF exhibited the highest. The oil absorption capacity (OAC) between the flour samples showed no significant (p < 0.05) difference, with CF documenting the highest value (1.83 g/g) and FF the lowest (1.72 g/g). The swelling power (SP) of the roasted flour samples (4.33 and 4.61 g/g) was significantly (p < 0.05) lower than that of unroasted CF (5.59%). The bulk density (BD) of flour is primarily influenced by its initial moisture content and starch content [p < 0.05) lower FC was observed in the evaluated roasted flour samples compared to the unroasted samples, whereas the foaming stability was significantly (p < 0.05) higher. Hence, roasted flour samples are incapable of forming dough.The data on the functional properties of the evaluated flour samples are shown in Table S7 in Supplementary Materials 2. The water absorption capacity (WAC) ranged from 0.66 to 1.61 g/g, with a significant  of 2%. Notably, the roasting treatment procedure either reduced or maintained the levels of LGC. The observed high gelling ability supports the usage of maize flour for porridge preparation , as wellee pasta .The goal of our research was primarily to investigate the quality characteristics of a specifically and traditionally processed maize flour and secondarily, to compare them with other similar flour products. The nutritional, functional, antinutritional, and antioxidant properties of Furniko flour (FF) were studied and compared to similar flour products. The findings of the study revealed that the traditional roasting process enhanced not only the nutritional content of the flour but also its functional properties and antioxidant potential. The FF appears to be rich in protein, fat, minerals, and phenolic compounds. Moreover, its functional properties and flavor profile make it an ideal candidate for the development of new food products. Our data support the common opinion among Pontic Greeks that FF is a healthy and nutritious food. Further research is necessary to support and introduce widespread consumption of FF’s healthy ingredients and organoleptic features.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Nuvol showed that our earlier species determination of Nuvolumbrosus Navás had been incorrect and that our “redescription” of the species actually applied to an undescribed species. Here, we redescribe the true N.umbrosus, based on a newly discovered male specimen. This specimen closely resembles Navás’ description, and it was collected from the Atlantic Forest as was the original type specimen. In addition, we assign the previously misidentified Nuvol specimens from the Amazonian region to a separate species, Nuvolsatur Sosa & Tauber, sp. nov. As a result of these actions, the genus Nuvol now contains two morphologically and geographically distinct species. In addition, the abdomens and genitalia of both sexes of Nuvol are now described .Examination of a newly discovered specimen of  Leucochrysini, a diverse and largely unstudied group in the neuropteran family Chrysopidae, currently contains ~190 described species uncomfortably classified into seven genera , as reported by The Neotropical green lacewing tribe n genera . One of  missing : 143. AlN.umbrosus Navás ;  Brotéria (Zoológica) 14: 25; “Rio de Janeiro, Febrero de 1912” (only one specimen). 19°53'2.86"S, 43°22'26.14"W, 751m] Peti, 30.iv.2012, A. F. Kumagai” , Belo Horizonte, Brazil).One male specimen preserved in alcohol, examined by FS: “MG, São Gonzalo Rio Abaixo, EA . Similarly, the linear alignment of the outer gradates and their flow into the PsM can be seen in most Leucochrysa (L.) species . Howevellus see , they ar


The extreme genetic and immunobiological heterogeneity exhibited by the African swine fever virus (ASFV) has been a significant impediment in the development of an efficacious vaccine against this disease. Consequently, the lack of internationally accepted protocols for the laboratory evaluation of candidate vaccines has become a major concern within the scientific community. The formulation of such protocols necessitates the establishment of a consensus at the international level on methods for the determination of homologous and heterologous isolates/strains of ASFV. The present article provides a comprehensive description of biological techniques employed in the classification of ASFV by seroimmunotypes. These techniques involve a holistic evaluation of ASFV isolates/strains based on their antigenic properties as determined by the hemadsorption inhibiting test (HAdI) using type-specific sera and an immunological test (IT) conducted on pigs inoculated with attenuated strains. The article outlines the methods for setting up the HAdI test, an IT on pigs, and the processes involved in the acquisition of type-specific serums for the HAdI test. It is pertinent to note that the definitive classification of seroimmunotype can only be ascertained after conducting an IT on pigs. The findings from the HAdI test or the phylogenetic analysis of the EP402R gene should be considered preliminary in nature. The course of the disease can be hyperacute, acute, subacute, and chronic . The caue family .African swine fever is currently having an unprecedented spread globally and one of the reasons for this spread is the lack of a safe and effective registered vaccine against the disease. It is known that pigs that have recovered after infection with the African swine fever virus (ASFV) can be protected from disease and/or death if subsequently infected with related virulent isolates/strains . In addiA widely used ASFV classification system is based on genotyping and determining the phylogenetic relationship of various isolates/strains. Currently, the studied isolates/strains of the ASFV are distributed into 24 genotypes . AlthougIn 1960, Malmquist and Hay were the first to document the occurrence of hemadsorption during the replication of ASFV in primary cultures of porcine bone marrow cells (PBMC) and primary peripheral blood leukocytes of swine (PBLS). In addition to the phenomenon of hemadsorption, they found that blood serum obtained from ASFV-infected pigs inhibits hemadsorption in ASFV-infected cultures of PBLS and PBMC cells, but does not affect the cytopathic effect and reproduction of the virus . Based oIn 1968, Coggins reported the isolation of non-hemadsorbing subpopulations of the ASF virus . FurtherThe seroimmunotype classification is based on the results of a comprehensive assessment of ASFV isolates/strains by antigenic properties in the HAdI test with type-specific sera and an IT inoculated with attenuated strains . The isoIn our opinion, it is important to establish unified international definitions for homologous and heterologous isolates/strains. Evaluation of candidate vaccines should be based on seroimmunotype classification of isolates/strains established in hemadsorption delay reaction and immunological tests. This article describes methods that are used for ASFV seroimmunotyping.The following terms will be used with the following meaning: serotype of ASFV is a group of isolates/strains of ASFV formed based on the results of the HAdI test; immunotype of ASFV is a group of isolates/strains of ASFV based on the results of IT; seroimmunotype of ASFV is a group of isolates/strains formed based on the results of grouping by HAdI test and IT.2.It is important to ensure that all plasticware used in the experiment is of tissue culture-grade quality. Additionally, all reagents and buffers should be properly sterilized either by autoclaving or filtration methods.2.1.2.1.1.In the experiments, use female or male pigs that are 3 to 4 months old, e.g., 30–40 kg piglets of the Large White Pig breed (Pigs). Place the animals in a BSL 3Ag laboratory. Conduct a 6-day acclimatization period before starting the study. Keep and euthanize pigs according to AVMA guidelines for the care and use of laboratory animals .2.1.1.1.2 incubator, refrigerator (4°C), analytical balance, pH meter, inverted microscope, centrifuge , and hemocytometer.Laminar flow cabinet, CO2.1.1.2.Vacuum blood collection tube, anti-coagulation , polypropylene conical tubes (50 mL), serological pipettes (10 mL), automatic pipettes with a volume of 20–200 μL, 100–1000 μL, aerosol-resistant filter tips, Millex-HV syringe filter unit, and 0.45 μm, PVDF .2.1.1.3.Complete blood leukocytes growing media (CBLGM): The Eagle’s minimal essential medium (EMEM) containing 10% of fetal bovine serum (FBS), penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2PO4, − 0.2 g, Na2HPO4 ×12 H2O – 2.9 g, and KCl – 0.2 g. distilled water to 1,000 mL. Check the pH before use. Store at 4°C.Phosphate-buffered saline pH 7.2, divalent cation-free (PBS): Nacl – 8.0 g, KH3 .Ficoll–Hypaque 1,077 g/cmDistilled water.Red blood cell (RBC) lysis buffer: 155 mM ammonium chloride, 12 mM sodium hydrogen carbonate, and 0.1 mM ethylenediaminetetraacetic acid. Sterilization by filtration.Trypan blue 0.4% (w/v).2.1.2.African swine fever virus productively infects monocyte/macrophage cells in the domestic pig. Here, we describe the culture of primary peripheral blood leukocytes of swine.Collect the required volume of fresh defibrinated pig blood into tubes containing an anticoagulant.Add 15 mL of Ficoll–Hypaque solution at 20–22°C to polypropylene conical tubes (50 mL) and overlay it with diluted pig blood in sterile PBS at a 1:1 ratio.Centrifuge the tubes at 1000×g for 30 min at 20–22°C with the brake off.Carefully aspirate the interphase layer using a serological pipette, transfer it to a new 50 mL tube, and then fill the tube with PBS to the desired volume.Centrifuge at 400×g for 10 min at 20–22°C.Remove the supernatant by carefully decanting or aspirating it, and then discard it appropriately. Add 5 mL of RBC lysis buffer to each tube, ensuring complete resuspension of the pellet, and incubate the tubes at 20–22°C for 5 min. After the incubation, add 40 mL of PBS and centrifuge the tubes at 400×g for 10 min at 20–22°C.Wash cells with PBS twice more. Resuspend cells in CBLGM at a volume 2.0–2.5 times greater than the initial blood volume.Mix 100 μL of the resuspended cells with 800 μL of PBS and 100 μL of trypan blue solution. Stir the mixture thoroughly. Proceed to count the number of clear (non-stained) cells using a suitable counting method, such as a hemocytometer. Based on the cell count, calculate the cell concentration using the appropriate formula or software. Adjust the volume of CBLGM cells to a concentration of 3.0–4.0 million cells/mL.2.2.2.2.1.Pigs (see 2.1.1).2.2.1.1.Laminar flow cabinet, refrigerator (4°C), bench-top centrifuge.2.2.1.2.Vacuum blood collection tube, anti-coagulation , 5 mL centrifuge tube with lid, serological pipettes (5 mL), automatic pipettes with a volume of 100–1000 μL, and aerosol-resistant filter tips.2.2.1.3.Complete blood leukocytes growing media: The EMEM containing 10% of FBS, penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2PO4, − 0.2 g, Na2HPO4 х12 H2O – 2.9 g, and KCl – 0.2 g. distilled water to 1000 mL. Check the pH before use. Store at 4°C.Phosphate-buffered saline pH 7.2, divalent cation-free: Nacl – 8.0 g, KH2.2.2.Centrifuge 1,0 mL of swine whole blood at 400×g for 5 min at 20–22°C.Remove plasma and buffy coat layer.Resuspend the red cells in 30 mL PBS and invert the tube to mix.Centrifuge the sample at 400×g for 5 min at 20–22°C and carefully remove and discard the supernatant. Repeat the washing step two more times using PBS, following the same centrifugation conditions.Resuspend RBCs in 1.0 mL of CBLGM, and then dilute the resuspended RBCs in a 1:100 ratio to prepare a 1% solution.2.3.2.3.1.Pigs (see 2.1.1), PBLS (see 2.1), ASFV strains seroimmunotype III: reference Mozambique-78, and attenuated MK-200.2.3.1.1.Laminar flow cabinet, refrigerator (4°C), freezer (− 40°C), water bath, and thermometer.2.3.1.2.Vacuum blood collection tube, clot activator , automatic pipettes with a volume of 20–200 μL, 100–1000 μL, aerosol-resistant filter tips, sterile 48-well Nunc cell culture plate with TC treatment, and 1.0 and 10.0 mL syringes.2.3.1.3.Complete blood leukocytes growing media: The EMEM containing 10% of FBS, penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2P(O)(OH)2] in sterile water (PAA).40% solution of phosphonoacetic acid [HOOCCH2.3.2.There are three main methods for obtaining sera that are active in the HAdI test from pigs: (i) survivors of ASF in subacute forms , (ii) pi50/mL; The infectious activities of ASFV strains were determined by titration in PBLS [four wells for each tenfold dilution; 2.3.2.1.2.0–103.0 HAU50.Infect pigs with a reference strain of ASFV of the selected seroimmunotype. Out of 10 pigs that are kept together, intramuscularly inoculate two pigs with a reference strain Mozambique-78 of ASFV of selected seroimmunotype III at a dose of 10The remaining eight animals would be infected by contact. When it is 14–21 days post the infection, examine the blood serum of survivors of the subacute form of ASF for the presence of active antibodies in the HAdI test (See 2.4.1).Obtain blood serum from surviving animals on day 42 after infection and test it in HAdI. Typically, their titers range from 1:40 to 1:10240.2.3.2.2.2.0 to 106.0 HAU50 does not induce the formation of hemadsorption-inhibiting antibodies. In some cases, natural and laboratory-obtained attenuated hemadsorbing strains that cause a chronic form of ASF induce the formation of HAdI antibodies in pigs with titers no higher than 1:20–1:80. The process of obtaining serums active in the HAdI test by the second method requires taking into account that, on the one hand, after infection with a virulent strain, immunized animals must survive, and on the other hand, they must get sick with the manifestation of characteristic clinical signs of ASF .2.3.2.3.The sodium salt of phosphonoacetic acid (phosphonate) inhibits the DNA polymerase of viruses by binding to the pyrophosphate site. Viral polymerase is significantly more sensitive to this drug than swine DNA polymerase. Phosphonoacetic acid, phosphonoacetic acid complex with 7-amino-1,3,5- triazaadamantane, and potassium pyridine salt of phosphonoacetic acid all prevented mortality of more than 80% of infected animals vs. 100% mortality of animals in the control group. The use of phosphonoacetic acid in combination with metisazone under microepizootic conditions prevented mortality of all piglets that had contact with the diseased ones, in comparison to 100% mortality in untreated animals. The possibility of obtaining type-specific sera was established, which removed the step of attenuation of virulent strains. The result allowed to shorten the serum preparation time by 3–12 times, which might be crucial for serotyping the virus since attenuation of individual strains can take up to 6–12 months or more .2.0 to 105.0 HAU50, with the specific virus dose adjusted based on the virulence of the ASFV strain.Administer intramuscular injection of the selected seroimmunotype III reference strain, e.g., Mozambique-78, to four pigs. The inoculation should be carried out at a dose ranging from 10Monitor the clinical signs and record temperature every day.Once the body temperature of the infected pigs starts to rise above 40°C and clinical signs of the disease become evident , begin administering intramuscular injections of a 40% PAA solution. The recommended dosage is 100–150 mg/kg of body weight. Administer the injections twice a day for the first 3 days, and then switch to once-a-day administration for the subsequent 10 days.On day 14, following the last recorded temperature peak, proceed to exsanguinate the animals and collect the blood samples. The collected sera should then be subjected to analysis using the HAdI assay.In the HAdI assay, the titer of anti-ASFV serum can range from 1:40 to 1: 640. Depending on the virulence of the ASF virus strain, mortality rates in affected animals can range up to 50%.In all cases, the selected type-specific pig serums are incubated for 30 min at a temperature of 56°C, aliquoted, and stored at −40°C. When selecting type-specific sera, it is assumed that their activity with homologous seroimmunotype reference strains of the ASFV in the HAdI test (see 2.4.1) should be from 1:40 or more (up to 1:10240), and with heterologous ones the inhibition of hemadsorption is not manifested.2.4.Serotyping of the ASFV in the HAdI test makes it possible to obtain a preliminary result on the seroimmunotype classification of the studied isolates relatively quickly, which saves money, effort, and time, compared to the conduction of an immunological test on animals. The HAdI test uses reference strains of nine ASFV seroimmunotypes and corresponding type-specific reference pig sera. There are two main modifications of the HAdI test formulation. The first one was proposed by 2.4.1.In the HAdI test, type-specific pig sera are used in working dilutions corresponding to their doubled titers. The titer of type-specific pig sera is taken as the highest dilution that causes inhibition of hemadsorption, multiplied by the dilution factor of the serum in the culture medium. The HAdI test in micropanel tablets is performed in the working volume, of which 0.90 is a cell suspension, 0.05 is a virus-containing material, and 0.05 is the serum of interest.2.4.1.1.PBLS (see 2.1), RBC (see 2.2), ASFV strain, Mozambique-78, swine anti-ASFV serums  to strain Mozambique-78, positive sera III serotype (PS), and normal porcine serum (NS) treated for 30 min at a temperature of 56°C.2.4.1.1.1.2 incubator, refrigerator (4°C), freezer (− 40°C), inverted microscope, and water bath.Laminar flow cabinet, CO2.4.1.1.2.Multichannel pipettes, automatic pipettes with a volume of 20–200 μL, 100–1000 μL, aerosol-resistant filter tips, and a sterile 48-well Nunc cell culture plate with TC treatment.2.4.1.1.3.Complete blood leukocytes growing media: The EMEM containing 10% of FBS, penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2PO4, − 0.2 g, Na2HPO4 ×12 H2O – 2.9 g, and KCl – 0.2 g. distilled water to 1,000 mL. Check the pH before use. Store at 4°C.Phosphate-buffered saline pH 7.2, divalent cation-free: Nacl – 8.0 g, KH2.4.1.2.Determination of working dilutions of investigated type-specific pig serums III serotypes from pigs (IS1 and IS2) in the HAdI test. The HAdI test is performed using 48-well plastic micropanels.2 incubator at 37°C for 3 days with 5% CO2 and 90% relative humidity.1. On day 0, prepare a suspension of PBLS with a concentration of 3.0–4.0 million cells/mL, and add 10 mL of the suspension to each well of the 48-well plates. Then incubate culture plates in a CO2. On day 3, take the following steps:2.5–103.0 HAU50/25 μL for the HAdI test.a. Calculate the dilution of viral stock required to achieve a concentration of 10b. Dilute the virus in CBLGM.c. Evaluate the status of adherent cells, specifically monocytes/macrophages, in the culture. Perform a wash by pipetting the medium up and down using an automatic pipette equipped with 1000 μL filter tips. This step aims to remove any non-adherent cells. Carefully discard the medium, and repeat the washing process two additional times to ensure thorough removal of non-adherent cells. Finally, add 450 μL of CBLGM to each well. Leukocyte culture should consist of single flattened, transparent cells uniformly distributed on the surface of the culture tablets with a density of at least 400–500 cells per field of view under low magnification .2.0–103.0 HAU50/25 μL/well. Leave the «cell culture controls» row not infected  of IS1, IS2, PS, and NS in CBLGM. Dispense 100 μL of CBLGM into 8 wells, occupying four rows (A-H) of the 48-well plate. In rows 1–4 , add 100b. Add 25 μL of the serum dilutions to the appropriate wells with infected cells.2 incubator.c. Incubate plates at 37°C for 2 h in a COd. To each well, add 50 μL of 1% swine RBC.2 incubator.e. Incubate plates overnight at 37°C in a COOn day 5, read hemadsorption using an inverted microscope. Note: the maximum dilution of each serum at which HAdI is complete (no rosetting cells) and write down the value. Monocytes/macrophages infected with ASFV with no anti-ASFV serum display a representative hemadsorption pattern. The result interpretation is represented in 2.4.2.The method of setting the HAdI test used at FRCVM differs from the method of The HAdI test for determining the serotype of ASFV isolates is carried out using the method described below, using 48-well plastic micropanels.2.4.2.1.6.0 to 107.5 HAU50/mL. In this study, we investigated the serotype classification of the Katanga-78 and Kaluga-20 isolates. Type-specific pig anti-ASFV serum of serotypes I-IX, with the WSD in the HAdI test not lower than 1:40 .PBLS (see 2.1). For serotyping purposes, the following reference strains of ASFV were utilized: I – Lisbon-57, II – Congo-49, III – Mozambique-78, IV – France-32, V – TSP-80, VI – TS-7, VII – Uganda, VIII – Stavropol 01/08, and IX – Davis. These viruses were propagated through 1–2 passages in cultures of PBLS cells, resulting in a viral titer ranging from 102.4.2.1.1.2 incubator, refrigerator (4°C), freezer (− 40°C), and inverted microscope.Laminar flow cabinet, CO2.4.2.1.2.Multichannel pipettes, automatic pipettes with a volume of 20–200 μL, 100–1000 μL, aerosol-resistant filter tips, and sterile 48-well Nunc cell culture plate with TC treatment.2.4.2.1.3.Complete blood leukocytes growing media: The EMEM containing 10% of FBS, penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2.4.2.2.2 incubator with a CO2 content of 5% and a relative humidity of 90%, and incubate at a temperature of 37°C for 3 days.1. On day 0, prepare a suspension of PBLS with a concentration of 3.0–4.0 million cells/mL. The working volume, 1.0 mL, of the prepared suspension of cells is introduced into each well of culture plates, and into each well of the 48-well plate. Culture plates in a CO2. On day 3, take the following steps:2.0–103.0 HAU50/25 μL for the HAdI test , PBLS (see 2.1).2.5.1.1.2 incubator, refrigerator (4°C), freezer (− 40°C), analytical balance, inverted microscope, and thermometer.Laminar flow cabinet, CO2.5.1.2.Multichannel pipettes, automatic pipettes with a volume of 20–200 μL, 100–1000 μL, aerosol-resistant filter tips, a sterile 48-well Nunc cell culture plate with TC treatment, and 1.0 mL syringes.2.5.1.3.Complete blood leukocytes growing media: The EMEM containing 10% of FBS, penicillin (100–200 IU/mL), and streptomycin (100–200 mg/mL).2.5.2.The infectious activity of the tested isolate, as well as the attenuated and virulent reference strains of the ASFV, is determined in the cultures of PBLS (See 2.3.2).6.0–107.5 HAU50.Pigs (eight heads) are inoculated twice, intramuscularly, with an interval of 14 days with attenuated ASFV strain MK-200 at a dose of 103.0 HAU50, and four with the studied isolate at the same dose. To control virulence, an additional two intact pigs each are infected in a similar way with a virulent reference strain of the ASFV and a test isolate at a dose of 103.0 HAU50.Then, after 28 days from the second inoculation, four pigs are intramuscularly infected with a virulent reference strain Mozambique-78 at a dose of 10During the experiment all pigs are monitored in terms of body temperature and other clinical signs of ASF. As a rule, during this time, the death of control animals is observed. The specificity of the disease and death from ASF of experimental pigs is confirmed by the isolation of the virus in the culture of PBLS.In the absence of death of at least 3/4 of pigs inoculated with an attenuated strain and subsequently infected with a virulent reference strain or a test isolate of the ASFV, they are considered to have an immunological correspondence and belong to the same seroimmunotype.2.5.2.1.In case the pigs inoculated with an attenuated strain and infected with a test isolate die from ASF, then this isolate does not have an immunological correspondence with the attenuated and virulent reference strains taken in the experiment. According to the existing classification, it should be assigned to the group of strains that do not match the results of serotyping in the HAdI test and in the immunological sample.If intact pigs infected with the tested isolate of the virus (virulence control) did not show signs of disease, characteristic symptoms, and did not die from ASF, this indicates its low virulence. After 21 days, these pigs are infected with a virulent reference strain of ASF virus of the presumed serotype. In case of survival of the pigs, the tested isolate of ASF virus is classified as belonging to the same serotype as the reference strain used.3.3.1.In 2015, a new approach was proposed that makes it possible to predict with a high degree of probability the seroimmunotype belonging to both hemadsorbing and non-hemadsorbing isolates of the ASFV . It is b3.2.The HAdI test is not applicable to determine the type of non-hemadsorbing ASFV isolates. Therefore, only an IT is used. If the non-hemadsorbing isolate is virulent, then its type affiliation is determined in an immunological test on pigs previously inoculated with attenuated strains of the ASFV. If the studied non-hemadsorbing isolates of the ASFV are avirulent or weakly virulent, then they are used on vaccinated pigs twice with an interval of 14 days; then, after 28 days pigs are infected with virulent hemadsorbing reference strains of the ASFV of various seroimmunotypes.3.3.The seroimmunotype classification has demonstrated its adequacy in studies on obtaining candidate live vaccines based on the selection of attenuated ASFV strains . AnotherIt should be noted that the final result of seroimmunotyping is achieved only as a result of setting an immunological test on pigs. Hemadsorption inhibiting test or phylogenetic analysis data on the EP402R gene should be considered preliminary. It was indicated above that the X group according to the seroimmunotype classification includes ASFV strains in which the HAdI results do not coincide with the IT . A possi3.4.3H-glycosamine-labeled proteins derived from lysates of PBM naturally susceptible A-cells infected with ASFV hemadsorbing strains as an antigen source; and (2) use of the homologous antisera with high activity in HAdI as an antibody source. Through extensive investigations, the serotype specificity of gp110-140 has been successfully determined . This glycoprotein exhibited a molecular weight ranging from 110 to 140 kDa and displayed a characteristic dumbbell-shaped band, which is typical for highly glycosylated proteins , 2018. Ftermined . While u3H-glucosamine labeled gp 110–140 preparations, derived from hemadsorbing ASFV reference strains purified with ion-exchange chromatography on DEAE-Sephacel, were used as antigens for the quantitative version of the radioimmunoprecipitation procedure. The percentage of specific binding obtained with the control sera of intact pigs was not greater than 3%. The serological relationship of gp110-140 with serotype-heterologous antisera varied from 20 to 45% which indicates that the gp110-140 contains both homologous and heterologous epitopes (A method exists for quantitative assessment of the serological relationship of hemadsorbing ASFV strains. Results of radioimmunoprecipitation are to be recorded not through a visual examination of the fluorogram, but by the number of pulses per minute using a β-counter. epitopes , 2018. Tin vitro and in vivo. It provides adequate experimental results during the development and evaluation of the protective properties of candidate vaccines against ASF. Knowledge about the seroimmunotype assignment of isolates/strains is useful in monitoring ASF, determining the phylogenetic relationships of virus isolates, and confirming the possible source of virus introduction.In summary, the described seroimmunotyping technique is based on the combination of two immunological tests The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The manuscript presents research on animals that do not require ethical approval for their study.AS: writing—original draft preparation. DK: conceptualization, supervision, and project administration. SN and VB: writing—review and editing. MV, IS, and GK contributed to both the conception and design of the work. All authors contributed to the article and approved the submitted version.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.


Pacific walruses are found in Arctic regions of the Chukchi and Bering Sea where rapid changes in environmental conditions resulting in loss of sea ice are occurring. Therefore, accurate life history data are crucial for species management plans and longitudinal data collected over the lives of individual walruses housed in zoos and aquaria provide otherwise difficult to obtain biological information.p < 0.0001) in weight (99 kg vs 57.6 kg/y) and length (26.9 cm vs 26.3 cm/y) with physical differences being detected by age 3 for weight and age 7 for length. Males reached adult weight at 13.5 ± 3.3 y and females by age 12.3 ± 2.3 y. The mean age at first ovulation and at first conception occurred at 8.8 y and 9.6 y. Greater than 75% of all conceptions and calving occurred between February and March and from May to June, respectively. Mean gestation lasted 423 d and false pregnancies lasted at least 169 d with a decrease (p < 0.05) in serum progesterone concentration between false pregnancy and pregnancy occurring within 6 months after ovulation. Based on these results, we estimated embryonic diapause to last from 120 to 139 days, and fetal growth last ~ 284 days. All males older than 8 y had an increase in serum testosterone and body weight that was highest in February and lowest in July. Overall, no differences were observed between male and female survival, with a mean (± SEM) life expectancy of 19.5 ± 1.5 y, respectively. Currently, the oldest male and female captive walruses are 40 and 43 y, respectively.While similar at birth, Gompertz regression curves indicated that males grew faster than females (Data provided herein include details of life history characteristics of zoo and aquaria housed walruses that are useful for wild population recovery models. In particular, results on survivorship and the identification of the most vulnerable period for calf survival can help with model development and suggests that for recovery to occur birthing locations for this species must be protected.The online version contains supplementary material available at 10.1186/s40850-022-00158-1. Odobenus rosmarus divergens) inhabits the Arctic and subarctic portions of the Pacific Ocean mostly along the edge of the sea ice in the Bering Sea during the winter and onshore during the summer in the Bering and Chukchi Seas   . For the= A(RG)] , 82. To = A(RG)] . If it w= A(RG)] , with IDp < 0.05. Once the optimum random variables were determined , all fixed effect variables were added to the model.To determine if seasonal monthly weight changes occur in non-pregnant adult females and to compare their weight against changes occurring during pregnancy the following parameters were defined. First, weight data (kg) were used only from mature females defined as ≥ 8 y. This was based on evidence that the majority of wild female walrus have ovulated by age 8 are thus considered mature , 13. Nexr = 0.989), and the recovery of known concentrations of standard added to a pool of sera was 86.8 ± 14.1% , thereby demonstrating negligible matrix interference. For progesterone, cross-reactivity of other hormones, according to the manufacturer’s directional insert, was: 17α-hydroxyprogesterone, 0.26%; 11-deoxycorticosterone, 0.81%; pregnelone, 0.49%; corticosterone, 0.001%; 5β-pregnan-3,20-dione, 0.06%; testosterone, 0.01%; cortisol, 0.002%; 5α-pregnan-3,20-dione, 3.78%; DHEA-S, 0.008%. For testosterone, parallel displacement of walrus sera compared to the standard curve was demonstrated (r = 0.998), and the recovery of known concentrations of standard added to a pool of sera was 99.5 ± 5.6% , For the testosterone assay, reported cross-reactivity, according to the manufacturer’s directional insert, was: androstenedione, 1.71%,; androsterone, 0.03%; aldosterone, not detected (ND); corticosterone, 0.01%; cortisol, ND; cortisone, ND; danazol, ND; 11-deoxycortisol, ND; dexamethasone, ND; DHEA, 0.1%; DHEA-S, ND; 5α-dihydrotestosterone, 0.81%; estradiol, ND; estrone, ND; ethisterone, 0.02%; fluoxymesterone, ND; 19-hydroxyandrostenedine, 0.04%; methyltestosterone, 0.15%; norethindrone, ND; prednisone, ND; progesterone, 0.02%; norethynodrel, 0.03%; spironolactone, 0.01%; triamcinolone, ND. The limit of detection, according to the manufacturer was 0.05 ng/ml for progesterone and 7 ng/dl for testosterone. Intra-assay variation for both hormones was < 10%. Inter-assay variation for three quality controls  was 14.4, 11.1 and 10.8%, respectively (n = 57) for progesterone and 9.9, 12.6 and 12.5% (n = 38) for testosterone. Routine internal quality control checks were performed daily, and sample reanalysis was performed whenever an outcome outside of the expected range for the respective stage of the reproductive cycle was observed.Serum progesterone and testosterone were analyzed using an immunoenzymometric assay . Assay validations for this species included sample parallelism and accuracy. Parallel displacement of walrus sera compared to the standard curve for progesterone was demonstrated  was difficult to ascertain due to the infrequency of sampling from animals after the breeding season, but to obtain an estimated length, we used the halfway point between last sample that had an above baseline progesterone (P) concentration (> 1 ng/ml) and the first sample that was continuously below baseline at the end of the FP period. Only samples that straddled these two P4 points and were separated by ≤ 60 d were used for this calculation. For example, the end of FP was determined to be the halfway point between two consecutive samples that were ≤ 60 d apart in duration, in which P4 concentrations decreased from above baseline to below baseline. Hormone concentrations in pregnant and FP females were then binned within months post ovulation , and for graphical display and discussion purposes month zero was assigned as March, the month the majority of conceptions occurred.p < 0.05. In addition to the dependent variable (progesterone), each model contained the appropriate fixed variables, month post conception (MPC), MPO, and/or status  and interactions . The continuous variable age was included in all models to account for any age associated changes in hormone production. The first analysis was conducted to compare for inter-monthly pregnancy progesterone concentrations aligned at month post-conception (MPC) across the entire pregnancy and a second one to compare monthly differences post-ovulation (MPO) between pregnancy and FP.For mean monthly hormone concentration comparisons within a pregnancy and between pregnancies, FP and non-pregnant, a 2 or 3 level REML was used with ID (level 2) and/or event ID  as the random variables as determined by estimated comparisons of models with and without the EID as the level 3 using the LR test and p < 0.05. For text, tables and graphs any transformed data were first back transformed, and then all data were presented as marginal means with 95% confidence intervals (CI) unless noted otherwise.All final mixed effects models were checked for normality using quantile plots of the standard residuals. If quantile–quantile plots of standardized residuals exhibited non-normal distribution the data were transformed as predicted by the Shapiro–Wilk test  until residuals were normalized. Multiple comparisons of marginal means were performed post-hoc using Sidak corrections at Kaplan–Meier modeling (KM) of daily survival rate for captive walruses was performed using animal life span data from the AZA inventory combined with data from SEA and KSW (duplicate entries removed) for all animals alive or added to facilities after January 1, 1980 until July 31, 2020 (Supplementary File Additional file 1. Additional file 2. Additional file 3.


To investigate the visual and patient-reported outcomes of a diffractive trifocal intraocular lens (IOL) in highly myopic eyes.Patients with planned cataract removal by phacoemulsification and implantation of a trifocal IOL (AT LISA tri 839MP) were enrolled in the prospective, multicenter cohort study. Patients were allocated into three groups according to their axial length (AL): control group, AL < 26 mm; high myopia group, AL 26–28 mm; extreme myopia group, AL ≥ 28 mm. At 3 months post-surgery, data for 456 eyes of 456 patients were collected, including visual acuity, defocus curve, contrast sensitivity (CS), visual quality, spectacle independence, and overall satisfaction.P < 0.001). In all three groups, about 60% of eyes achieved uncorrected near and intermediate visual acuity of 0.10 logMAR or better, but significantly fewer eyes in the extreme myopia group achieved uncorrected distance visual acuity of 0.10 logMAR or better (P < 0.05). Defocus curves revealed that the visual acuity was significantly worse in the extreme myopia group than others at 0.00, − 0.50, and − 2.00 diopters (P < 0.05). CS did not differ between the control and high myopia groups but was significantly lower in the extreme myopia group at 3 cycles per degree. The extreme myopia group also had greater higher-order aberrations and coma, lower modulation transfer functions and VF-14 scores, more glare and halos, worse spectacle independence at far distance, and consequently lower patient satisfaction than others .After surgery, the uncorrected distance visual acuity improved from 0.59 ± 0.41 to 0.06 ± 0.12 logMAR (In eyes with a high degree of myopia (AL < 28 mm), trifocal IOLs have been shown to provide similar visual outcomes to those in non-myopic eyes. However, in extremely myopic eyes, acceptable results may be obtained with trifocal IOLs, but a reduced level of uncorrected distance vision is expected.The online version contains supplementary material available at 10.1186/s40662-023-00336-3. To meet the increasing visual demands of patients, multifocal intraocular lenses (IOLs) are widely implanted during cataract surgery to achieve spectacle independence , 2. BifoIn the last two decades, the prevalence of high myopia has increased dramatically worldwide . PatientHowever, there have been few studies on the effectiveness of trifocal IOLs in highly myopic eyes, partly because trifocal IOLs are mostly used in European countries where the prevalence of high myopia is relatively low . One potThis prospective multicenter cohort study was initiated by the Eye and Ear, Nose, Throat (EENT) Hospital of Fudan University, Shanghai, China, between October 1st, 2019 and October 1st, 2021, in collaboration with three other medical institutions: Xinshijie Zhongxing Eye Hospital, Shanghai Aier Eye Hospital, and Shanghai Bright Eye Hospital in Shanghai, China. The protocol was approved by the institutional review board of EENT Hospital of Fudan University (No. 2020109), and the study was conducted in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients.The inclusion criteria were cataract patients aged ≥ 20 years, with a corneal astigmatism of ≤ 1.0 diopter (D), a mu chord of ≤ 0.4 mm, and a willingness for spectacle independence. The exclusion criteria included high corneal higher-order aberration (HOA) > 0.5 μm, zonular weakness, strabismus, severe retinal pathology, uveitis, glaucoma, previous ocular procedure or trauma, occupation incompatible with dysphotopsia, severe psychiatric disorder, or any systematic disease that affected visual acuity, such as diabetes. According to the definition of high myopia and previous studies , 14, theBefore surgery, all patients underwent complete ophthalmic examinations, which included visual acuity, slit-lamp examination, corneal topography , AL measurement , and a fundus examination. The IOL power required was calculated with the Barrett Universal II formula (Lens Factor + 1.62 or A Constant 118.5) and was selected with a target of emmetropia –16. No mwww.zeiss.com). The ophthalmic viscosurgical device was thoroughly removed before the incision was hydrated. The position of the IOL was checked before the completion of surgery. No intraoperative complication was observed in the study.Surgery was performed by an experienced surgeon in each medical institution  using standard procedures. After making a 2.6 mm clear corneal incision temporally, a continuous curvilinear capsulorhexis with a diameter of approximately 5.5 mm, was created. After cataract removal by phacoemulsification, the anterior chamber was filled with an ophthalmic viscosurgical device. The trifocal IOL  was then implanted in the capsular bag with the injection technique recommended by the manufacturer. The IOL power ranges from 0.0 D to + 32.0 D with 0.5 D increments  at 40 cm, uncorrected intermediate visual acuity  at 80 cm, uncorrected distance visual acuity  at 4 m, and corrected distance visual acuity  were evaluated using an Early Treatment Diabetic Retinopathy Study (ETDRS) chart  under 85 cd/m2) or mesopic conditions (background luminance of 3 cd/m2). Patches not seen were accounted with the value of the last patch, and all patients saw at least one patch. The log base 10 contrast sensitivity values were used for statistical analysis according to previous studies [Monocular contrast sensitivity (CS) was assessed with best distance correction using the Optec6500 . Assessments were made at spatial frequencies of 1.5, 3, 6, 12, and 18 cycles per degree (cpd), with or without glare, under photopic  and the modulation transfer function (MTF) were measured with an OPD Scan III aberrometer  , 19, 20 To assess their subjective visual quality, all patients completed a visual function questionnaire (VF-14). VF-14 is scaled from 0 to 100, with a higher score indicating better subjective visual function. The patients were also asked about symptoms of dysphotopsia, including glare, halos, and starbursts. Four-point response scales were used to assess each symptom: from “never” to “always” for the frequency, from “none” to “severe” for the severity, and from “not bothered at all” to “bothered very much” for bothersomeness. Information on spectacle independence and overall satisfaction was collected by asking the patients whether they used spectacles for near vision , intermediate vision , or far vision  and whether they were satisfied with the visual outcomes after surgery.t-test was used to compare preoperative and postoperative visual acuity. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to compare continuous variables among groups. The χ2 test was used to compare categorical variables among groups. All statistical analyses were performed with SPSS software  and missing data were not imputed. All tests were two-tailed, and the level of statistical significance was set to P < 0.05.Means ± standard deviations were used to describe continuous variables, and percentages were used to describe the distributions of categorical variables. A paired Adverse events are included in Fig. P > 0.05) other than AL and calculated IOL power.The mean age of all the participants was 59.4 ± 11.1 years, and 56.6% were female. The mean AL of the highly myopic eyes was 27.78 ± 1.39 mm . Table t-test; P < 0.001). In all three groups, about 60% of eyes achieved UNVA and UIVA of 0.10 logMAR or better  and CDVA . Table P > 0.05), but the mean UDVA and CDVA were significantly worse in the extreme myopia group than in the control group .After trifocal IOL implantation, UDVA improved significantly in all patients, from 0.59 ± 0.41 logMAR before surgery to 0.06 ± 0.12 logMAR at three months after surgery (paired P > 0.05). The percentage of eyes with SE of more than 1.0 D was significantly higher in the extreme myopia group than in the control group . The percentage of eyes with a prediction error of within ± 0.5 D did not differ among the control, high myopia, and extreme myopia groups , but the percentage of eyes with a prediction error of more than 1.0 D was significantly higher in the extreme myopia group than in the control group .No difference in the spherical equivalent (SE) and prediction error was detected among the groups .The monocular defocus curves for the trifocal IOLs are shown in Fig. P > 0.05). However, the extreme myopia group showed significantly worse photopic and mesopic CS at 3 cpd without glare compared with the control and high myopia groups . When measured with glare, the extreme myopia group also showed significantly worse photopic CS at 1.5–6 cpd and significantly worse mesopic CS at 1.5–3 cpd .Monocular contrast sensitivity under photopic and mesopic conditions with or without glare are shown in Fig. P = 0.019 and 0.015, respectively). The extreme myopia group also showed significantly greater intraocular HOAs and coma aberration than the other two groups . At a pupil diameter of 4 mm, the ocular and intraocular HOAs and coma aberrations were significantly greater in the extreme myopia group than in the control group . The extreme myopia group also displayed significantly lower ocular and intraocular MTFs than the other groups at spatial frequencies of 10, 20, and 30 cpd, for both pupil diameters .The HOAs and MTFs of the trifocal IOL in different groups are presented in the Additional file P = 0.007). 2.2% (6/273), 1.9% (2/105) and 6.4% (5/78) of the control, high myopia and extreme myopia group had great difficulties driving at night from the VF-14, respectively . Glare was more frequent in the extreme myopia group than in the control group , although there was no difference in its severity among the three groups , although there were no differences in the severity or bothersomeness of halos among the groups. The frequency, severity, and bothersomeness of starbursts did not differ among the groups .In terms of subjective visual quality, the extreme myopia group had a significantly lower VF-14 score than the other two groups .Table 2 test, P = 0.041). Six patients in the control group and two patients in the extreme myopia group felt dissatisfied with the outcome of surgery. Their reasons for dissatisfaction were mainly spectacle dependence for near and far vision, poor CS, and significant symptoms of dysphotopsia. Of the high myopia group who were partly satisfied, one patient showed high refractive error, and two others cannot tolerate dysphotopsia. Of the extreme myopia group who were dissatisfied, one showed high refractive error, and another showed poor CS.The rates of patient satisfaction in the control, high myopia, and extreme myopia groups were 91.6%, 97.1%, and 87.2%, respectively  as in eyes without high myopia. However, in extremely myopic eyes, acceptable results may be obtained with trifocal IOLs, but a reduced level of uncorrected distance vision is expected due to the higher prediction error in IOL power calculation. Despite this, the implantation of multifocal IOLs in patients with AL > 28 mm is not contraindicated, as long as patients are informed about the potential visual disadvantages before surgery. Further research is needed to assess the long-term safety of trifocal IOLs in this population.Additional file 1: Figure S1. Higher-order aberrations (HOAs) and modulation transfer functions after implantation of the trifocal intraocular lens. Ocular and intraocular HOAs for 6 mm  and 4 mm  pupil diameters. Ocular and intraocular modulation transfer functions (MTF) at different spatial frequencies for 6 mm  and 4 mm  pupil diameters. RMS, root mean square; cpd, cycles per degree. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001.