Prevention of myopia should begin before school age. However, few population-based cohort studies have investigated refractive status in preschool children with cycloplegia. This study aimed to investigate the post-COVID-19 refraction and ocular biometric parameters of preschool children in Beijing Tongzhou District.A population-based cohort study of kindergarten children in Tongzhou District, Beijing, commenced in November 2021. The present study reports data from the first year of the aforementioned population-based study. We selected children aged 3–6 years from nine kindergartens. Biometric parameters, including axial length (AL), anterior chamber depth (ACD), and corneal radius of curvature (CR), were collected before cycloplegia. Cycloplegic refraction was also measured. The spherical equivalent (SE), lens power (LP), and AL-to-CR ratio were calculated. Multiple linear regression analysis was used to analyse the correlation between refraction and ocular biometric parameters.A total of 1,505 children completed the examination, and a mean SE of 1.24 ± 0.91 D was found. The overall prevalence of myopia was 1.93%. The mean AL, ACD, CR, LP, and AL-to-CR ratio were 22.24 ± 0.70 mm, 3.28 ± 0.26 mm, 7.77 ± 0.26 mm, 26.01 ± 1.56 D, and 2.86 ± 0.07, respectively. Longer AL, deeper ACD, larger AL-to-CR ratio, and lower LP were associated with older age; the CR was not significantly different among different ages. In the multiple linear regression analysis, after adjusting for sex and age, the model that included AL, CR, and LP explained 87% of the SE variation. No differences were observed in the prevalence of myopia or the SE in this particular age range.The findings of this study suggest that a large proportion of preschool children in Beijing are mildly hyperopic, with a considerably low prevalence of myopia. In preschool children, refractive development was found to present mild hyperopia rather than emmetropia or myopia, a phenomenon that is characteristic of this age range. The process by which human eyes transition from hyperopia to emmetropia is often called emmetropization . Both geFew population-based cohort studies have investigated preschool children with cycloplegia. Guo et al.  found thTo understand the post-COVID-19 refraction and ocular development in preschool children, we initiated a population-based cohort study in Tongzhou District, Beijing. From 2021, the refractive changes and their influencing factors in these preschool children will be obtained via follow-up. This article reports the first-year results of this study.This is a population-based cohort study conducted from November 2021 in kindergartens in Tongzhou District, Beijing. Tongzhou District is a suburban area and serves as the sub-center of Beijing, with economic and population levels falling in the middle range among Beijing’s districts. There are a total of 181 preschools in Tongzhou District, comprising 38 public preschools and 153 private preschools. Using a stratified random cluster sampling method, we selected 9 schools, including 2 public preschools and 7 private preschools, with a total of 1917 participants. Among them, 185 individuals were not permanent residents of Tongzhou District and were excluded from the study due to the difficulty of follow-up. Consequently, invitations to participate were extended to 1732 individuals. Among the invited individuals, 439 potential participants were from public preschools, and 1293 potential participants were from private preschools. The two selected public preschools had 150 and 289 potential participants, respectively, while the seven private preschools had 112, 121, 140, 150, 214, 271, and 285 potential participants each.The project conformed to the tenets of the Declaration of Helsinki and obtained ethical approval from the Ethics Committee of Beijing Tongren Hospital Affiliated to Capital Medical University (TRECKY2020-152). Before the examination of each kindergarten, we held an online parents’ meeting to explain the purpose and methods of the study, as well as the precautions for cycloplegia. We also obtained the informed consent. A total of 1,515 (87.5%) individuals, with consent forms signed by their parents or legal guardians, participated in the study.The research team consisted entirely of ophthalmologists, optometrists, and eye nurses, each of whom underwent standardised training. The distant vision test was performed using Lea Symbols 3-m Set charts  to accommodate children’s cognitive ability. Refraction before and after cycloplegia was measured using an autorefractor machine . Each eye was measured three times and averaged. The spherical difference between any two measurements should be < 0.5 D; otherwise, additional measurements were taken. Ocular biometry was performed before cycloplegia using the Lenstar 900 . Each eye was measured three times, and each measurement was automatically checked for quality by the instrument. If a measurement was deemed unsatisfactory, additional measurements were conducted. The average of the three tests was calculated as the results. Anterior segment and strabismus examinations were performed by an ophthalmologist.Cycloplegia was induced by 1% cyclopentolate. Each child was given one drop of 0.5% proparacaine hydrochloride in each eye, followed by two drops of 1.0% cyclopentolate  5 min apart. After 30 min, if the pupil size was ≥ 6 mm and the light reflex was absent, cycloplegia was deemed adequate. Otherwise, an additional drop of cyclopentolate was given, and if the above standard of pupil size and light reflex had not been reached after 15 additional minutes, failure of cycloplegia was recorded.The spherical equivalent (SE) was equal to spherical diopters (D) + 0.5 × cylindrical diopters (D). Myopia was defined as an SE of ≤ − 0.50 D, emmetropia was defined as 0.50 D < SE < 0.50 D, mild hyperopia was defined as 0.50 D ≤ SE < 2.00 D, and mild-to-high hyperopia was defined as an SE of ≥ + 2.00 D. The corneal radius (CR) was equal to the mean of the flattest and the steepest radius. The axial length (AL)-to-CR ratio was calculated as AL divided by the CR. The lens power (LP) was based on the Bennet–Rabbetts  formula,SAS 9.3 software was used for data processing and analysis. An independent samples t-test was used to compare the ocular characteristics, including SE, AL, ACD, CR, AL-to-CR ratio, and calculated LP, between boys and girls. Trend analysis was used to examine the age-related differences. When normal distribution was not met, the Mann-Whitney test was used for comparisons. Multiple linear regression analysis was used to analyse the correlation between refraction and age, sex, and ocular biometry parameters of the children. There was a strong correlation between the left and right eyes (r = 0.87), and only the right eye was included in the statistical analysis. P < 0.05 was considered statistically significant.x2 = 0.0084, P = 0.93). Among them, 793 were boys (52.7%). Moreover, 208, 507, 659, and 131 were aged 3, 4, 5, and 6 years, respectively. The average age was 4.97 ± 0.78 years. No statistically significant differences in sex, age, or ethnicity were noted between the included and excluded groups . Mild hyperopia (0.50 ≤ SE < 2.00 D) was the predominant refractive state, and the proportion of children with mild hyperopia increased with age (Ptrend = 0.012). The proportion of children with mild-to-high hyperopia (SE ≥ + 2.00 D) decreased with age (Ptrend = 0.017). The proportion of children with emmetropia and myopia was minimal, and no age differences were noted . Myopia was rare in this population, with a rate of 3.05% in children aged 6 years. Figure The refractive-state distribution across various ages is shown in Table trend < 0.001), with measurements increasing from 21.96 ± 0.64 mm at 3 years of age to 22.60 ± 0.68 mm at 6 years of age. Boys had significantly longer ALs than girls aged 3–6 years (P < 0.001). The ACD increased from 3.20 ± 0.29 mm at 3 years of age to 3.40 ± 0.21 mm at 6 years of age (Ptrend < 0.001). ACD was deeper in boys than in girls, with statistically significant differences across all age groups. No statistically significant difference was found in CR among the different age groups during the preschool stage, with an average CR of 7.77 ± 0.26 mm. Except for 3-year-olds, boys exhibited slightly larger CR values than girls, implying that the boys’ corneas were flatter than those of the girls.The age distribution of the ocular biological parameters is shown in Table trend < 0.001). Boys had a slightly greater ratio than girls, except for 3-year-olds; nonetheless, the overall difference was negligible. The LP was calculated using the Bennett–Rabbetts formula, with a mean value of 26.01 ± 1.56 D, and demonstrated a significantly decreasing trend from 27.33 ± 1.54 D at 3 years of age to 25.07 ± 1.31 D at 6 years of age (Ptrend < 0.001). Girls had a greater LP than boys at all ages.The mean AL-to-CR ratio was 2.86 ± 0.07, increasing from 2.82 ± 0.06 to 2.89 ± 0.07 from 3 to 6 years of age   and ShenSE, spherical equivalent; AL axial length, CR, corneal radius of curvature.Our study found that among children aged 3–6 years of age, the older they were, the longer the AL, the deeper the ACD, the greater the AL-to-CR ratio, and the smaller the LP. These findings are consistent with previously reported developmental patterns in school-aged children, marked by axial elongation and LP reductions , 27. TheThe AL-to-CR ratio is an effective indicator of myopia, and the optimal diagnostic cutoff value is reportedly > 2.99 . The AL-The effectiveness of AL-to-CR ratio in evaluating the SE was statistically superior to that of AL, explaining 40% of the SE variance. Linear regression demonstrated that the offset to myopia was 8.79 D for every one-unit increase in the AL-to-CR ratio. When AL, CR, and LP were included in the predictive calculation model, they explained 87% of the SE variance. AL and LP were negatively correlated with the SE. We observed fluctuations in the mean SE between the ages of 3 and 6. The SE value at age 3 was + 1.18 diopters. Subsequently, there was a gradual increase to + 1.29 diopters at age 4, followed by a subsequent decrease to + 1.19 diopters at age 6. However, in this study, no age difference in the SE was noted, a phenomenon that may be due to the limited age range of 3–6 years; nonetheless, a decrease in LP is known to compensate for the longer AL with increasing age, preventing the development of myopia in children –33.However, our study has certain limitations. First, LP was calculated instead of being measured. Rozema demonstrated that LP calculated using the Bennett–Rabbetts formula was comparable to that measured using the alphabet method in adults with emmetropia and myopia . HoweverWe described the first-year data from a cohort study on refraction and ocular biometric parameters in preschool children in Beijing. In this population, the majority had hyperopia, while the proportion of those with myopia was substantially low. With age, mild-to-moderate hyperopia gradually shifted toward mild hyperopia but did not progress to myopia, exhibiting consistency with the results of several studies and potentially elucidating the process of refractive development in children. Changes in the refractive statuses of preschool children in Beijing will be observed upon follow-up.


Enterobacter asburiae named E7 was isolated from the intestine of healthy common carp and displayed an extensive antibacterial spectrum against Aeromonas hydrophila, A. veronii, A. caviae, A. media, A. jandaei, A. enteropelogenes, A. schubertii, A. salmonicida, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. E7 was nonpathogenic to the host and susceptible to the majority of antibiotics used in human clinical practice. E7 could grow between 10 and 45°C and between pH 4 and 7 and was extremely resistant to 4% (wt/vol) bile salts. Diets were supplemented with 1 × 107 CFU/g E. asburiae E7 for 28 days. No significant difference in the growth of fish was observed. Expression of immune-related genes IL-10, IL-8, and lysozyme in common carp kidney was significantly upregulated at weeks 1, 2, and 4 (P < 0.01). A significant upregulation of IL-1β, IFN, and TNF-α expression was observed after week 4 (P < 0.01). There was a significant increase in mRNA expression of TGF-β at week 3 (P < 0.01). Following challenge by Aeromonas veronii, the survival rate (91.05%) was significantly higher than observed in the controls . Collectively, E. asburiae E7 is a promising new Gram-negative probiotic that can enhance health and bacterial resistance of aquatic animals and could thus be developed as an exclusive aquatic probiotic.Probiotics are an alternative strategy for antibiotics, but most probiotics are Gram-positive bacteria suitable for terrestrial animals. Therefore, it is imperative to develop dedicated probiotics for the common carp industry to be ecologically efficient and environmentally friendly. A novel IMPORTANCE In the present study, we evaluated for the first time the efficiency of Enterobacter asburiae as a prospective probiotic for aquaculture applications. The E7 strain showed extensive resistance to Aeromonas, no pathogenicity to the host, and stronger environmental tolerance. We observed that the resistance of common carp to A. veronii was enhanced by feeding a diet containing 1 × 107 CFU/g E. asburiae E7 for 28 days, but growth was not improved. Strain E7 can act as an immunostimulant to induce the upregulation of some innate cellular and humoral immune responses, resulting in enhanced resistance to A. veronii. Hence, the continuous activation of immune cells can be maintained by adding suitable fresh probiotics to the diet. E7 has the potential to act as a probiotic agent for green, sustainable aquaculture and aquatic product safety. Lactococcus lactis TW34, Carnobacterium sp. T4, and L. pentosus H16 isolated from Patagonian fish effectively decreased the counts of Vibrio and Enterobacter (Enterobacter sp. G87 protected seabass larvae and Artemia nauplii from Vibrio harveyi infection (Bacteria are the most common pathogens in farmed fish and cause significant losses in both freshwater and marine aquaculture –3. Antim–robacter . Zakarianfection .Cyprinus carpio) belongs to the order Cypriniformes and the family Cyprinidae, which is regarded as the largest family of freshwater fish. Common carp are considered a potential source for commercial aquaculture in many countries because of its high adaptability to the environment and food (–Aeromonas species are the main cause of motile Aeromonas septicemia (MAS) in fish and are widely found in freshwater environments  as live microorganisms that can enhance vitality, strengthen immunity, promote digestive system function, and confer health benefits to the host when consumed in suitable doses . A cruciobiotics . There aEnterobacter asburiae by morphology, physiology and biochemistry, molecular biology, and bioinformatics. In vitro antimicrobial activity, biosafety, and physical tolerability were evaluated. In addition, the aim of this study was to evaluate the potential of E. asburiae E7 as a feed probiotic for growth, immunity, and resistance to A. veronii infection in common carp.In the current research, a promising probiotic strain named E7 was isolated from healthy common carp intestines and identified as in vitro antagonism assay. Twenty-seven strains were antagonistic to common aquatic pathogens (Enterococcus agar (EA), Pfizer Enterococcus-selective agar (PSE), freshwater fish agar (FWA), Bacillus megatherium medium (BM), tryptone soy agar (TSA), brain heart infusion (BHI) and MRS medium, but a small numbers in MRS. The growth curve of E7 in LB broth medium is shown in Fig. S1 in the supplemental material.A total of 151 strains of bacteria were isolated and purified from the intestines of 10 healthy common carp for the following athogens , with onathogens . The strThe colonies formed after 24 h of incubation at 28°C were 2 to 4 mm in diameter and had typical colony characteristics, including pale yellow or milky white in color and round with a smooth surface and regular edges . This baEnterobacter genus. A maximum likelihood (ML) phylogenetic tree based on 16S rRNA gene sequences revealed that strain E7 did not cluster into any branch . In addition, an ML phylogenetic tree of the gyrB gene sequences revealed that strain E7 closely clustered with E. asburiae , no significant differences in survival within 7 days compared to the control group .PCR coupled with agarose gel electrophoresis showed that E7 was missing most of the ARGs, but E. asburiae E7 was found to be viable at pH 4 and grew well quantitatively and morphologically under experimental culture conditions at pH values of 5 to 7 (f 5 to 7  and D. If 5 to 7  and E. Ff 5 to 7  and F.E. asburiae E7 diet administration on the growth performance of common carp are shown in E. asburiae) decreased slightly compared with the controls, but the difference was not statistically significant.The effects of P < 0.05) (P < 0.05) (P < 0.01), while GSH activity was significantly upregulated at 28 days (P < 0.05) (P < 0.01)   and tota < 0.05)  and tota < 0.05)  than the < 0.05) . Analogo < 0.01) .IL-1β), interleukin-10 (IL-10), interleukin-8 (IL-8), interferon (IFN), tumor necrosis factor-α (TNF-α), lysozyme, and transforming growth factor-β (TGF-β).The relative mRNA expression of immune-related genes in the kidneys of common carp fed with a diet containing strain E7 is displayed in IL-1β, IFN, and TNF-α were observed to be significantly upregulated after week 4 of feeding (P < 0.01)  (TGF-β (P < 0.01)  , and E.  < 0.01) , and F.  < 0.01)  between A. veronii to assess the protective efficacy of E. asburiae E7 against bacterial infection in fish, and the survival curves are presented in 7 CFU/g E7. Statistical analysis demonstrated that dietary supplementation with strain E7 significantly enhanced the resistance of common carp to A. veronii infection (P < 0.01)  .Bacillus cereus var. toyoi isolated from soil decreased the mortality of Japanese eel infected with Edwardsiella sp. Likewise, supplementation in the feed improved the growth rate of yellowtail , has a critical role in DNA replication and is widely distributed in bacterial species  analyses, morphological, physiological, and biochemical characterization identified the isolate E7 as E. asburiae.Comparative analysis of the 16S rRNA sequence is the most common genotype identification method for bacteria . Strainsy branch . In comp species . GeneralRNA gene , so the  species . In the ae clade . Cultureae clade . The stre et al. . Coupledin vitro antagonism assays with pathogens that were exposed to candidate probiotics or their extracellular products  and psychrophilic nonmotile Aeromonas . In addition, E7 could also antagonize other common fish pathogens, such as Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, and Shewanella. So far, no similar results have been reported for E. asburiae. According to some researchers, the antibacterial property of probiotics may be related to nutrition competition or inhibitory substances produced by bacteria, such as broad-spectrum antibiotics, bacteriocins, organic acids, siderophores, hydrogen peroxide, and enzymes  of fish after 28 days of E7 feeding. In addition, the biological and ecological security of potential probiotic strains is crucial in the selection process. Consequently, strains should be tested for resistance to various common antibiotic classes  and subsequent confirmation that resistance genes or virulence plasmids have not been transmitted  and source. In terms of security and effectiveness, probiotics from hosts are far better than those screened from other environments . To ensuSparus auratus helped to promote growth and improve stress tolerance to high stocking density  cells, and lymphocytes or by activating lymphocytes during tissue damage and pathogen invasion , Pfizer Enterococcus selective agar (PSE), MRS agar (MRS), freshwater fish agar medium (FWA), Bacillus megatherium medium (BM), brain heart infusion agar medium (BHI), and tryptone soy agar (TSA), and cultivated at 28°C for 24 h. Different single colonies were picked randomly according to different phenotypes. The isolation bacterial sampling process was repeated to acquire pure colonies. These colonies were picked with a sterile inoculum needle and inoculated on LB plates coated with pathogenic indicator bacteria. Following incubation at 28°C for 24 h, antibacterial activity was shown in the inoculated circles around the inoculation site, which was an important reference for selecting isolates. The strains with antimicrobial activity were preserved for further study.Strain E7 used in the present research was isolated from the healthy common carp gut. Intestinal tissues from healthy common carp were collected under sterile conditions, rinsed with sterile saline (0.85%), and subsequently homogenized with sterile saline. The bacterial solution was diluted 1,000-fold with sterile saline. The diluted suspension (0.1 mL) was then spread on eight different kinds of medium agar plates, such as Luria-Bertani agar (LB), gyrB were amplified by PCR using the following primers: 16S rRNA gene, 27F forward primer (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492 R reverse primer (5′-TACGGTTACCTTGTTACGACTT-3′), and gyrB, forward primer (5′-GAAGTCATCATGGTTCTGCAYGCNGGNAARTTYGA-3′) and reverse primer (5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′). The PCR conditions were as follows: 1 cycle at 95°C for 5 min, followed by 32 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s, and 1 cycle at 72°C for 10 min. The PCR products were sequenced  to obtain nucleotide sequences and were subjected to similarity analysis and multiple sequence alignment by NCBI BLAST. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences was performed for accurate identification of the strain. The purified strains identified were then stored in 50% (wt/vol) glycerol at −80°C for subsequent studies.Bacteria were identified by physiological and biochemical tests and Gram stained according to the manufacturer’s instructions . Subsequently, genomic DNA of candidate strains was extracted using a genomic DNA kit . The sequences of the 16S rRNA gene and A. hydrophila, A. media, A. veronii, A. jandaei, A. caviae, A. enteropelogenes, A. schubertii, A. salmonicida, Vibrio alginolyticus, V. cholerae, Pseudomonas aeruginosa, Ps. putida, Plesiomonas shigelloides, Acinetobacter johnsonii, Shewanella sp., and Citrobacter braakii) and the strains from gut were incubated in LB broth medium at 28°C for 24 h at 160 rpm. Subsequently, 0.1 mL of pathogen culture diluted to a concentration of 1 × 107 CFU per mL by colony counting method was plated on LB agar plates. After all bacterial solutions were absorbed by agar plates, sterile paper discs of 6 mm in diameter containing 10 μL of the isolated suspension (1 × 107 CFU/mL) were placed on the plate and coincubated at 28°C for 24 h. Antibacterial activity was detected by the transparent area around the inoculated paper. All aquatic pathogenic bacteria used in this study were supplied by The Fisheries Disease Lab of Northwest A&F University.Sixteen common aquatic pathogenic bacteria  to detect hemolysin. The activated strain E7 was punctured on a blood agar plate with an inoculating needle and incubated at 28°C for 24 h. Hemolysis was determined according to the following hemolysis types. Alpha-hemolysin production results in the decomposing red blood cells and a green ring formed around the colony. Beta-hemolysin forms a clear ring around the colony. No transparent loops or green areas were regarded as nonhemolytic. 9 CFU/mL) and sterile phosphate-buffered saline  to observe the survival rate within 7 days.Twenty fish were equally divided into two groups, then each fish was injected intraperitoneally (i.p.) with 0.1 mL of strain E7 suspension (1 × 107 CFU/mL) was coated on LB agar plates, and discs containing 35 medical antibiotics  and 11 aquatic/veterinary antibiotics were placed on the surface of LB agar and incubated for 24 h at 28°C. The diameters of zones of inhibition were measured, and the antimicrobial susceptibility was divided into susceptible (S), resistant (R), and intermediate resistant (IR) according to the diameter of inhibition circle.The sensitivity of E7 strains to antibiotics was evaluated by the paper diffusion method. Strain E7 , three tetracycline resistance genes , two quinolones resistance genes (qnrA and qnrS), two chloramphenicol resistance genes (cmlA and floR), two β-lactam resistance genes (blaTEM and blaCMY), one macrolide resistance gene (ermA), and one aminoglycoside resistance gene (aac[6′]-Ib) . The total PCR volume was 20 μL, consisting of 10 μL of 2× Taq master mix, 8 μL of double-distilled water (ddH2O), 10 pmol/L forward and reverse primers, and 1.0 μL of bacterial genomic DNA. After initial enzyme activation at 94°C for 10 min, 32 cycles of amplification  were performed, followed by extension at 72°C for 10 min. The PCR product was analyzed by electrophoresis in a 1.5% agarose gel for 40 min at 100 V and 50 mA.PCR was used to determine whether the bacteria carried ARGs. The quality and concentration of the amplified products were evaluated by agarose gel electrophoresis (1.5% agarose in 1× Tris-acetate-EDTA [TAE] buffer) and UV. Fourteen common ARG types in water environments were selected, including three sulfonamide resistance genes (7 CFU/mL) was inoculated at 1% (vol/vol) in LB broth medium with different pH values and bile salt concentrations, which were incubated at 28°C and 160 rpm for 24 h. Activated strain E7 was inoculated at 1% (vol/vol) into LB broth medium and incubated at 35, 40, 45, 50, 55, 60, 65, and 70°C for 24 h in a thermostat water bath to determine temperature tolerance. The absorbance at a wavelength of 600 nm was measured using 0.2 mL of the above bacterial culture. Meanwhile, 0.1 mL of bacterial culture was coated on LB solid medium and cultivated at 28°C for 24 h. Photographs were taken, and bacterial tolerance was recorded.Tolerance to pH, temperature, and bile salts was assessed for E7 based on previous studies, with some modifications . LB brotE. asburiae E7 was obtained using the above isolation methods and was determined to be nonpathogenic by in vitro hemolysis assay and in vivo injection assay and could be used for subsequent diet supplementation. The E7 isolate was inoculated onto LB agar plates using bacterial inoculators for pure culture, and single colonies were inoculated into LB broth medium for proliferation at 28°C for 24 h. The concentration was calculated by the colony counting method after 10-fold gradient dilution of the E7 culture suspension with sterile PBS.Cyprinus carpio) weighing 0.57 ± 0.06 g were acquired from Guangzhou Menghu fish seed farm . Fish were housed in tanks (400-L water capacity) at 23 to 25°C for 30 days before the start of the experiment and were adapted to laboratory conditions . Fish were fed at 2% of body weight twice every day (8:30 and 16:30). The water in the experimental tank was replaced one-third of the way through every day. The water used in the study was aerated 24 h in advance and adjusted to the optimum temperature.Common carp , with 3 replicates of 50 fish in each group. Different diets were fed to each group: three tanks were added without the base diet (control group), and the other three tanks were provided with E7 at 1 × 107 CFU/g of the strain diet. Each tank represented one biological repetition per group.Three hundred fish of similar sizes were randomly divided into two groups  for subsequent RNA extraction.To reflect the macroscopic effect of E7 on common carp growth, 15 fish were randomly selected from each replicate every 7 days to determine body weight and length, and 6 were picked to measure carcass weight (excluding viscera weight).Serum was obtained once every 7 days to measure acid phosphatase (ACP) activity, alkaline phosphatase (AKP) activity, total protein level (TP), glutathione level (GSH), total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) activity. Measurements were performed using commercial kits from Nanjing Jiancheng Institute following the manufacturer’s instructions.actin gene was used as a reference gene. The PCR cycling conditions were:1 cycle at 95°C for 180 s and 40 cycles at 95°C for 20 s, 60°C for 30 s, and 72°C for 20 s. Melting curves and analyses were performed as described in Li et al.  were stained with hematoxylin and eosin (H&E) and examined using a light microscope equipped with an imaging system (Olympus BX53).A. veronii in LB broth for 24 h at 28°C, the culture was diluted to obtain 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 CFU/mL suspensions. Every 10 common carp were i.p. injected with 0.1 mL of the above-mentioned bacterial suspension with different dilution concentrations, and the results revealed that the 7-day 50% lethal dose (LD50) of A. veronii was 9.857 × 106 CFU/mL ; thus, the concentration used for the challenge test was approximately 1 × 107 CFU/mL. A formal test was then performed. After 28 days of feeding, 0.1 mL of a suspension of A. veronii (1 × 107 CFU/mL) was injected i.p. into each fish from experimental and control groups (n = 50). Survival rate  × 100 was used to access the protective efficacy of the E7 strain against A. veronii. Pathological changes were observed daily for 8 days, and the dead fish were removed promptly.Pretests were performed before the challenge. After incubation of t tests or a Mann-Whitney U test, respectively. All results are presented as the mean ± standard deviation (SD). Single asterisks  and double asterisks  indicate statistical significance compared with control. A P value of <0.05 and a P value of <0.01 were regarded as statistically significant and extremely significant, respectively.Statistical analyses and drawing of all data were performed using GraphPad Prism 8.0 . Normally and nonnormally distributed data were compared using independent-samples gyrB sequences of E7 are available in public databases with accession numbers OP522325 and OP558470.The 16S rRNA and


Three-dimensional matrices are a new strategy used to tackle type I diabetes, a chronic metabolic disease characterized by the destruction of beta pancreatic cells. Type I collagen is an abundant extracellular matrix (ECM), a component that has been used to support cell growth. However, pure collagen possesses some difficulties, including a low stiffness and strength and a high susceptibility to cell-mediated contraction. Therefore, we developed a collagen hydrogel with a poly (ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN), functionalized with vascular endothelial growth factor (VEGF) to mimic the pancreatic environment for the sustenance of beta pancreatic cells. We analyzed the physicochemical characteristics of the hydrogels and found that they were successfully synthesized. The mechanical behavior of the hydrogels improved with the addition of VEGF, and the swelling degree and the degradation were stable over time. In addition, it was found that 5 ng/mL VEGF-functionalized collagen/PEGDA IPN hydrogels sustained and enhanced the viability, proliferation, respiratory capacity, and functionality of beta pancreatic cells. Hence, this is a potential candidate for future preclinical evaluation, which may be favorable for diabetes treatment. Type I diabetes mellitus T1D) is a chronic metabolic disease characterized by the destruction of pancreatic beta cells D is a ch,3. ThereSome studies have reported that extracellular matrix (ECM)-based matrices can sustain islet and beta cell growth and functionality ,9,10,11.Type I collagen is an ECM protein abundant in many tissues, including the pancreas . Hence, Several strategies to improve collagen hydrogel stiffness, strength, and resistance to degradation and cell-mediated compaction have been developed using different cell models, including poly(ethylene glycol) diacrylate (PEGDA) ,17,18. TIn the current study, we developed a collagen hydrogel (2 mg/mL) with a covalently crosslinked PEGDA network to increase the mechanical response by adding VEGF at different concentrations  to evaluate the effect of the degree of functionalization on the hydrogels. We analyzed the physicochemical characteristics of the biomaterials and their rheological behavior in response to deformation. We established the swelling behavior and its degradation over time. Thereafter, we evaluated the effects of the developed hydrogels on the viability, growth, mitochondrial respiration, and functional behavior of beta pancreatic cells. The aim of our study was to develop a collagen hydrogel with a PEGDA IPN functionalized with VEGF directly loaded to mimic the pancreatic environment for the sustenance of beta pancreatic cells as a strategy for diabetes therapy.−1 positions were observed, representing OH bonds, carbonyl groups, and NH2. These bonds are strongly related to amide A of Type I collagen, especially when crosslinked [2 at 2870 cm−1, which corresponds to amide B found in natural collagen [FTIR elemental analysis was performed to demonstrate the integration of the basic components into the chemical structure of the functionalized collagen/PEGDA IPN hydrogels . Initialsslinked ,27. Carbsslinked ,27. We acollagen . Neverth−1, bonding between NH and the C=O stretch formed by amide I, which governs the secondary structure of the peptides, was observed. [−1 indicates C–N stretching and N–H bending vibrations, which correspond to amide II and are consistent with type I collagen [−1, confirming that the intact triple-helical structure of collagen was well maintained [At 1640 cmbserved. ,27 Additcollagen . We obseintained ,31. Previntained ; however−1, which corresponded to the asymmetric stretching of CH2 and the symmetric vibration of C=C groups, respectively, typically found in the PEGDA backbone [−1 is usually associated with the C=O of the acrylate groups in PEGDA [−1, which are associated with the C–O–C and C–O vibrational modes of the PEGDA backbone, were also observed. This suggests that PEGDA acts as an interpenetrating network (IPN) in the collagen matrix, and PEGDA was successfully integrated. Notably, the photoinitiator aids the polymerization process by the homolytic bond cleavage of radicals upon exposure to UV light [−1 band has been associated with the VEGF peptide, as previously suggested [In particular, there was an overlap at 2870 and 1640 cmbackbone ,34. Nonein PEGDA ,36. MoreUV light . Similaruggested . Furtherp = 0.0021) and 5 ng/mL (p = 0.0163) had a significantly larger pore distribution than the 2 mg/mL concentration. These pores varied for the 3 ng/mL condition from 1.818 to 9.993 μm and for the 5 ng/mL condition from 1.110 to 8.615 μm.To evaluate the microstructural modification of the functionalized collagen/PEGDA IPN hydrogels, scanning electron microscopy (SEM) was performed, as shown in This analysis suggests that high concentrations of VEGF induced structural changes in the microstructure of the collagen/PEGDA hydrogel because larger pores were found under the 3 and 5 ng/mL experimental conditions. Our findings are consistent with some reports in which VEGF-loaded structures induced the presence of pores . These sWe performed a rheological analysis in order to evaluate the storage (𝐺’) and loss (𝐺’’) moduli of functionalized collagen/PEGDA IPN hydrogels as a function of the shear stress applied . It is sInterestingly, VEGF-loaded hydrogels exhibited a higher elastic behavior than non-functionalized constructs. However, increasing the VEGF content did not affect the stiffness of the functionalized hydrogels. This may be explained by the direct loading method used to functionalize the constructs. Hence, the hydrogel becomes crowded, and the VEGF–collagen interaction increases; thus, the elastic behavior increases . HoweverAll hydrogels presented a linear viscoelastic region (LVE), which expresses the independence between 𝐺’ and 𝐺’’ of the deformation that occurs. This is directly related to the stable and structured shapes of the hydrogels ,42. The In addition, p > 0.05), but there was a tendency for the 3 and 5 ng/mL VEGF conditions to have a greater water absorption capacity than the 2 mg/mL and 1 ng/mL VEGF conditions. This characteristic is a key parameter because a high degree of swelling makes them excellent candidates for highly biocompatible materials [To determine the water absorbency capacity and in vitro degradability over time, the swelling ratio and degradation percentage were assessed . On day aterials .p > 0.05) in the swelling ratio for the 2 mg/mL, 1 ng/mL, and 3 ng/mL VEGF hydrogels, with mean values of 12.847 ± 0.500, 12.767 ± 1.437, and 14.907 ± 2.034, respectively. These results are consistent with those of Munoz-Pinto et al. [After eight days of culture, we found that the 5 ng/mL VEGF condition had a high swelling ratio (16.077 ± 1.178). However, there was a decrease . However, it is important to highlight that we found that the 5 ng/mL hydrogel had a percentage of over 95%, whereas the other conditions ranged from 90 to 93%. This is consistent with previous findings, where collagen hydrogels were found to retain a large water volume owing to the high hydrophilicity of the collagen peptide [To verify this, we analyzed the water content percentage B and fou peptide ,16,17. F peptide . Hence,  peptide .In contrast, we evaluated the percentage of in vitro degradation over time C. All hyAdditionally, we evaluated the release profile of VEGF as a function of time . For 3 aThe main mechanisms mediating the release of this protein are the swelling and degradation rates of the hydrogels, as previously suggested ,51,52. ATo evaluate the viability and proliferation of encapsulated beta pancreatic cells in collagen/PEGDA IPN hydrogels, we performed live/dead, MTT, and PicoGreen assays . The livIn addition, we performed SEM to analyze the distribution of beta pancreatic cells and their interactions with the collagen/PEGDA IPN matrix. As shown in p > 0.9999), 98.204 ± 21.299 (p > 0.9999), and 103.907 ± 11.207% (p > 0.9999), respectively, compared to CCP. However, after the same time period, 5 ng/mL of VEGF showed a significant (p = 0.0028) increase in cell viability (151.957 ± 5.582%) compared to CCP, suggesting that cell growth was stimulated. This was confirmed by a cell proliferation assay (p < 0.001), 252.300 ± 7.093 (p < 0.001), and 256.000 ± 0.750% (p < 0.001), respectively.Additionally, we confirmed the ability of these hydrogels to support live beta pancreatic cells using the MTT assay C. After on assay D after 7Previous reports have found that collagen/PEGDA IPNs can sustain cell viability, but cells do not proliferate ,17. A poThe increased proliferation of beta pancreatic cells in functionalized hydrogels may be due to the mesopores that are influenced by VEGF, as shown in the SEM analysis in Another possible explanation may be the presence of VEGF, as other reports have stated that this growth factor helps the formation of islets, and, thus, cell proliferation ,58. Addip > 0.05). However, basal respiration was improved at a concentration of 5 ng/mL of VEGF, with a mean value of 40.577 ± 4.586 pmol/(s × 106 cells) (p = 0.0183) compared with the 2 mg/mL condition.To evaluate oxygen flow in beta pancreatic cells and the influence of hydrogels on these cells, we performed high-resolution respirometry . After 2p = 0.4390), 9.254 ± 3.216 (p = 0.9204), 9.889 ± 0.571 (p = 0.9688), and 16.418 ± 2.517 (p = 0.6436) pmol/(s × 106 cells), respectively, compared to CCP.No significant differences were observed in the leak state between the experimental groups. In this respiratory state, we found that 2 mg/mL and 1, 3, and 5 ng/mL of VEGF had mean oxygen flow values of 6.184 ± 2.849 (p = 0.0068) and 48.888 ± 6.669 (p = 0.0278) pmol/(s × 106 cells), respectively, compared to CCP. Similarly, under 3 and 5 ng/mL VEGF conditions, the uncoupled state was not affected. The oxygen flow in the uncoupled state for 3 ng/mL was 53.157 ± 4.425 (p = 0.0729) and for 5 ng/mL was 62.737 ± 3.383 (p = 0.5004) pmol/(s × 106 cells) compared to CCP.In the state of maximal respiration capacity in the uncoupled state, we found that 2 mg/mL and 1 ng/mL of VEGF significantly decreased oxygen consumption with mean values of 42.487 ± 1.932 , 1.409 ± 0.083 (p < 0.0001), 1.281 ± 0.046 (p = 0.0011), and 1.457 ± 0.075 μIU/mL × μg DNA (p < 0.0001), respectively, compared to CCP. Similarly, we stimulated the encapsulated cells with a high glucose concentration. At this concentration, all experimental groups showed an increase in insulin secretion, with mean values of 1.574 ± 0.037 (p = 0.0019) for 2 mg/mL, 1.520 ± 0.029 for 1 ng/mL (p = 0.0082), 1.497 ± 0.117 for 3 ng/mL (p = 0.0151), and 1.687 ± 0.048 for 5 ng/mL (p = 0.0002), for μIU/mL × μg DNA, respectively.We performed glucose-stimulated insulin secretion analysis (GSIS) to evaluate the effect of the functionalized collagen/PEGDA IPN hydrogels on the functional behavior of beta pancreatic cells . After 4p = 0.0246) when compared with CCP. Nevertheless, the secretion index was sustained in the functionalized collagen hydrogels .However, we found that the insulin secretion index B . Physicochemical analysis of the hydrogels confirmed the presence of the characteristic elemental groups of type I collagen, along with the presence of PEGDA and VEGF. In addition, we determined that hydrogels with PEGDA and VEGF exhibited conformational changes in the biomaterial matrix, which may have influenced the stable water retention and weight loss. Furthermore, we found that the hydrogels were tunable and mechanically stable with a gel-like solid structure. We performed biocompatibility, cell respiration, and functionality assays, which helped us to confirm that almost all functionalized hydrogels were able to sustain or improve these parameters. However, our results cumulatively indicated that the 5 ng/mL VEGF treatment resulted in an increased cell viability, improved proliferation, sustained oxygen consumption, and enhanced insulin secretion. To the best of our knowledge, this is the first study to successfully synthesize a functionalized collagen/PEGDA IPN hydrogel that supports and induces the growth of functional beta pancreatic cells without affecting mitochondrial respiration. This is a promising biomaterial that may be used in future preclinical studies on diabetes treatment in clinical beta cell replacement therapy.2, according to the manufacturer’s guidelines. The cells were maintained in a RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS) , 2 mM of Glutamine , and penicillin/streptomycin/amphotericin B 1% . The medium was changed every three days and subcultured with trypsin/EDTA , at a density of 2 × 105 cells/cm2.The BRIN-BD11 (ECACC 10033003) cell line was cultured at 37 °C-5% CO6 cells/mL and microdispensed in a 96 well-plate. After 5 min of incubation at 37 °C, the hydrogels were treated with PEGDA 10%  and photoinitiator 0.1%  solutions and allowed to infiltrate the collagen network for 30 min at 37 °C. PEGDA within the collagen network was crosslinked by 5 min of exposure to longwave UV light , and an interpenetrating network (IPN) was formed. The collagen/PEGDA IPN hydrogels were incubated in the culture medium at 37 °C and 5% CO2.Collagen/PEGDA IPN hydrogels were fabricated at a collagen concentration of 2 mg/mL and functionalized with vascular endothelial growth factor (VEGF) at different concentrations ,23. In b−1 with a resolution of 4 cm−1 and eight scans. The spectra obtained were plotted using OriginLab Pro Software Version 9.8.5 .FTIR spectroscopy was performed to analyze the functional chemical groups and successful inclusion of components in the developed hydrogels . The dehMorphological features of the functionalized collagen/IPN hydrogels with and without cells were analyzed by scanning electron microscopy (SEM). Briefly, the IPN hydrogels were rinsed and fixed in 4% paraformaldehyde . Louis, MO) and 2.5% glutaraldehyde . They were treated with an increasing series of ethanol concentrations   and hexamethyldisilazane .Samples were sputter-coated, and micrographs were acquired using a Field Emission Gun (FEG) QUANTA FEG 650 with a secondary electron detector  at an accelerating voltage of 15 kV and magnifications of 6000 × and 15,000×.i), and incubated for 8 days at 37 °C in media cultures. The final swollen mass (ms) was weighed and dehydrated to determine the final dry mass (md). The mass swelling ratio (q) was calculated as follows for each condition (1):Swelling and degradation tests were performed to assess the capacity of the collagen/PEGDA IPNs to absorb water and their stability over time, respectively. These were used as indicators of the degree of infiltration within the collagen networks and the effect of VEGF on the hydrogels ,46,66. HThe water content (%) is defined by Equation (2):The degradation test was performed using Equation (3):Rheological behavior was analyzed to determine the deformation and flow of the hydrogel. This analysis was performed using an MCR 302 Anton-Paar Rheometer  with 20 mm diameter parallel plates and a Peltier system for temperature control (37 °C). Amplitude sweeps tests were performed, and a shear strain of 0.1–300%, angular frequency of 10 rad/s, and frequency sweeps of deformation of 0.1% for all samples were applied to determine the storage (𝐺’) and loss moduli (𝐺’’) of the samples.An in vitro release study was performed to evaluate VEGF release. VEGF peptide contains tryptophan, tyrosine, and cysteine residues, whose ability to absorb light at 280 nm is commonly used to determine the concentration of proteins in solution . Hence, 2 atmosphere. The constructs were rinsed with PBS 1× and DMSO was added to dilute the formazan crystals formed. The absorbance at 570 nm was measured using a Multiskan GO spectrophotometer . The quantitative findings were microscopically confirmed using the live/dead assay with Calcein AM and ethidium homodimer-1  and incubated for 45 min at room temperature. Fluorescence imaging was performed using a fluorescence microscope Olympus BX53 . Images were processed using the ImageJ software .To assess the effect of functionalized collagen/IPN hydrogels on the cell viability of pancreatic beta cells, we performed 3-2,5-diphenyltetrazolium bromide  and live/dead assays . Briefly® dsDNA Kit  [To evaluate the effect of the hydrogels developed on cell proliferation, DNA content was measured using the Quant-iT™ PicoGreenDE, USA) . Briefly6 cells/mL for each condition were incubated for 24 h and then placed in a chamber with the culture medium at 37 °C under gentle agitation (350 rpm). The oxygen flow was evaluated at different respiratory states: basal, leak, and uncoupled [6 cells)) and expressed as relative values compared to cell culture plastic (CCP).The mitochondrial respiratory capacity of the hydrogels was analyzed using a high-resolution Oxygraph-2K spectrometer . Cells encapsulated in hydrogels at a concentration of 2×10ncoupled . Basal r2, 1.2 mM of MgSO4, 1 mM of NaH2PO4, 22 mM of NaHCO3, 10 mM of HEPES, and 0.1% BSA ). The supernatant from each condition after exposure was collected and insulin secretion was measured using a Rat Insulin ELISA kit . Values were normalized to DNA content (μg/mL) using the Quant-iT PicoGreen assay. Finally, the stimulation index (SI) was calculated as the ratio of high- to low-glucose insulin secretion.To assess beta cell functionality, a static glucose-stimulated insulin secretion (GSIS) assay was performed . Brieflyp < 0.05 was considered significant . Each experiment was repeated at least three times.Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism, Version 7 . One-way analysis of variance (ANOVA) was performed followed by a post hoc Tukey’s test or Fisher’s LSD, where


New psychoactive substances continue to appear on the drug market, and alpha-pyrrolidinoisohexanophenone (α-PiHP) is one of the most popular cathinone derivatives. In this article, we report a case of death caused by α-PiHP. Based on the toxicological results of the studied case along with autopsy, histopathological findings and crime-scene information, fatal intoxication with α-PiHP was accepted as the final cause of death. α-PiHP and its metabolite (OH-α-PiHP) were detected and quantified in all postmortem materials . To date, this is the first case of determination of α-PiHP and its metabolite in postmortem specimens. In our opinion, α-PiHP and its metabolite concentration database can be helpful in the interpretation of fatal cases. According to the Polish Sanitary Inspector in his warning on 11 July 2022 , 4. The A 37-year-old man  was agitated and acting strangely, and then he lost consciousness. Witnesses called an ambulance, but after a resuscitative action, the doctor declared the man dead. The man was addicted to drugs. A bag of white powder labeled “α-PiHP” was found next to the corpse. The autopsy was performed by a forensic pathologist ∼48 h after death. The autopsy revealed cerebral edema and congestion, subconjunctival and subpleural petechial hemorrhages, pulmonary edema and congestion, low-grade atherosclerosis, liver enlargement, traces of rescue operations, fluidity and stagnation of blood in internal organs. The histological examination showed high-grade cerebral edema, brainstem edema, cerebellar edema and hyperemia, diffuse cardiac scarring, recent ischemic foci of myocardial fibers, cardiomyocyte hypertrophy, focal pulmonary edema and hyperemia, nodular goiter, hyperemia and chronic interstitial nephritis, chronic non-specific portal space inflammation in the liver, splenic congestion, pancreatic autolysis and fibrosis, increased congestion and colitis, erosive hemorrhagic gastritis and adrenal congestion. For toxicological analysis, samples of blood , vitreous humor, cerebrospinal fluid, cerebral cortex, brainstem, cerebellum, bile, liver, kidney, heart, pancreas, spleen, thyroid, lung, adipose tissue, stomach and intestine were collected during the autopsy. The collected samples were stored at −20°C until analyses, which were performed within 5 days from the autopsy.3 at a concentration of 0.1 mg/mL was chosen as the internal standard (IS) and was acquired from Sigma-Aldrich . All the remaining reagents used in the analyses were of high purity grade (high-performance liquid chromatography (HPLC) and mass spectrometry grade). Acetonitrile, 35% ammonia solution, ammonium formate, ethyl acetate, formic acid, methanol and water Chromasolv were acquired from Sigma-Aldrich .α-PiHP at a concentration of 1 mg/mL in methanol was acquired from Chiron . Mephedrone-d3 spiking solution was prepared for use as the IS to obtain a final concentration of 100 ng/mL in each body fluid sample and 100 ng/g in homogenates of each of the tissues. Together with the analyzed samples, calibration samples were prepared. Calibration samples were obtained through the addition of the appropriate volumes of the working solutions to 0.2 mL of each body fluid to obtain the final concentrations of 20, 50, 100, 200, 500, 1,000 and 5,000 ng/mL and 0.2 g of each homogenized tissue to obtain the final concentrations of 100, 1,000 and 5,000 ng/g (n = 3 for each level).To quantify α-PiHP in the autopsy specimens, the standard addition method was employed. Mephedrone-d3 as the IS, including calibration samples, 15% ammonia (pH 12) and 0.5 mL acetonitrile were added. The mixtures were briefly vortexed and ultrasonicated for 60 min. Then, to each sample, 2.0 mL of ethyl acetate was added. Finally, the test tubes were shaken for 2 h and centrifuged, and the supernatant layer was collected and evaporated to dryness in a stream of nitrogen at ambient temperature. The dry residues were dissolved in 0.5 mL methanol and the samples were transferred to the glass inserts placed in the vials and they were ready for the analyses using liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS-MS).The liquid–liquid extraction procedure was used to prepare biological samples. To 0.2 mL of each body fluid containing mephedrone-d3 as the IS, including calibration samples, was minced and homogenized. The extraction procedure was the same as described earlier.A 0.2 g of each tissue containing mephedrone-dA 10-mg sample of the white powder was dissolved in 1 mL of methanol, ultrasonicated for 10 min and then centrifuged. The resulting solution was further diluted 1:100 v/v with methanol. This diluted sample extract was transferred to the glass insert and vial and then analyzed using gas chromatography–mass spectrometry (GC–MS).m/z 50–500.For the routine qualitative screening of α-PiHP in biological samples, the LC–MS screening method for illicit drugs and new psychoactive substances (NPSs) was used. Extracts were analyzed using HPLC ACCELA connected to a linear ion trap mass spectrometer Finnigan LTQ . Chromatographic separation was performed using a Hypersil GOLD C18  analytical column . The mobile phase was composed of Solvent A (0.02 M aqueous solution of formic acid and 0.05 M aqueous solution of ammonium formate) and Solvent B (10% Solvent A and 90% acetonitrile). The injection volume was 10 μL. The flow rate was 0.5 mL/min. A 40-min gradient was used, changing the composition as follows: 5% Solvent B (0.00–2.00 min) and 60% Solvent B (2.00–32.00 min), where it remained isocratic for 2.00 min. It was followed by re-equilibration for 6 min. Samples were ionized in the electrospray ionization (ESI) mode with positive ionization, and the ion monitoring range was The confirmatory analysis of α-PiHP in biological samples was carried out using LC–MS-MS multiple reaction monitoring. Extracts were analyzed using ultra-high performance liquid chromatography Dionex UltiMate 3000 liquid chromatograph  connected to a TSQ Endura mass spectrometer . The chromatograph was equipped with an RP-MS Accucore column . The mobile phase was composed of Solvent A (0.02 M aqueous solution of formic acid and 0.05 M aqueous solution of ammonium formate) and Solvent B (10% Solvent A and 90% acetonitrile). The injection volume was 10 μL. The flow rate was 0.5 mL/min. A 14-min gradient was used, changing the composition as follows: 10% Solvent B (0.00–0.50 min) and 95% Solvent B (0.50–7.00 min), where it remained isocratic for 1.00 min. It was followed by re-equilibration for 6 min. Samples were ionized in the ESI mode with positive ionization. The LC–MS-MS screening method had been validated for α-PiHP and OH-α-PiHP. Tandem MS conditions are presented in For the GC–MS analysis of the white powder, the Thermo Trace 1310 gas chromatograph  coupled with the ISQ LT single quadrupole mass spectrometer  was used. The analyses were carried out with the use of the Rxi®-5Sil MS column . The following working parameters were employed: injector temperature, 260°C; oven temperature, 100°C for 2 min, ramp at 20°C/min to 260°C; carrier gas (helium) flow rate, 1.2 mL/min; MS transfer line temperature, 250°C; MS source temperature, 250°C; and injection volume, 1 μL, on splitless mode. The obtained mass spectra in the positive electron impact ionization (EI) mode were compared with the spectra from the Cayman Chemical EI mass spectra library (2022) .The results obtained were processed with the use of the Xcalibur v. 4.0. program.The validation data for the LC–MS method for α-PiHP are presented in the m/z 246) were detected in all body fluids and tissues, which was responsible for the death of a man. The presence of α-PiHP metabolite (m/z 248) was also detected in all body fluids and tissues. The molecular formula of this metabolite corresponded to a reduction of the β-ketone moiety in α-PiHP to the corresponding alcohol metabolite. The product ion m/z 230 was obtained in the MS2 (tandem) mode from the precursor ion m/z 248, indicating the elimination of one water molecule from the protonated molecular ion [M + H − H2O]+ . This transformation is a well-known feature of the cathinones. The second product with m/z 173 suggested a water loss and a dealkylation. The proposed fragmentation pattern of α-PiHP and OH-α-PiHP is presented in As a result of the performed analyses, high concentrations of α-PiHP m/z 46 were dC/P). However, in some circumstances, using the ratio of xenobiotic concentration in the liver to the peripheral blood (L/P) or both factors is more accurate. New redistribution assessment coefficients are being sought. When the C/P ratio is <1.0 , the drug is not prone to redistribution, but resuscitation attempts may result in a C/P ratio <1.0  in postmortem human specimens. This is the first study that presented the distribution of α-PiHP and its metabolite OH-α-PiHP in postmortem biological fluids and tissues from cases. The concentrations of metabolites combined with the parent drug concentration can be helpful in the interpretation of fatal cases involving α-PiHP and contribute knowledge to the scarce data reported in the international literature. The detection of relevant metabolites in addition to the parent drug may be useful for other researchers and scientists working in the field of clinical and forensic toxicology. The presence of α-PiHP metabolite in biological specimens can be crucial for indisputably proving the ingestion of the drug, so the abuse or intoxication with α-PiHP may be provable unequivocally. The presented case also shows that the evaluation of toxicological results along with histopathological alterations and circumstantial information is fundamental in determining the correct cause of death.bkad026_SuppClick here for additional data file.


BackgroundTotal hip arthroplasty (THA) is the most successful orthopedic elective surgical procedure for end-stage hip arthritis. THA is linked with significant blood loss, ranging from 1,188 to 1,651 mL, and a transfusion rate of 16-37%, which frequently results in postoperative blood transfusions. Postoperative blood transfusions can be avoided by using autologous blood transfusion, intraoperative blood saving, local anesthetic, hypotensive anesthesia, and antifibrinolytic medications such as tranexamic acid (TXA) administration.MethodologyA double-blinded, placebo-controlled, randomized, controlled study was conducted with three prospective groups to investigate the efficacy of topical and systemic routes of a single intraoperative dose (1.5 g) of TXA. Patients were recruited from our center between October 2021 to March 2022 who were undergoing primary total hip replacement. Estimated blood loss was calculated and compared in groups, and a p-value of <0.05 was taken as significant.ResultsA total of 60 patients were recruited in our study. Estimated blood loss was similar in both treatment groups, 816.8 ± 219.9 mL in the systemic TXA group and 775.5 ± 107.2 mL in the topical TXA group. The placebo group had 1,066.3 ± 150.4 mL estimated blood loss, which was significantly higher compared to the treatment groups.ConclusionsAdministration of TXA (1.5 g) significantly lowers blood loss without increasing problems, which can eliminate concerns about intravenous TXA use. TXA reduces blood loss by 270 mL on average. Total hip arthroplasty (THA) is one of the most successful orthopedic elective surgery for end-stage hip arthritis . THA hasThree prospective groups participated in the study to examine the effectiveness of topical and systemic delivery of a single intraoperative dose 1.5 g) of TXA. For the systemic TXA, local TXA, and placebo groups, the allocation ratios were 1:1:1. Patients receiving primary total hip replacements were included from our center between October 2021 and March 2022. Patients were not included if they had a history of thrombosis or a risk of developing it, refused blood transfusions, had a known allergy to TXA, had an active thromboembolic disease, were pregnant or nursing, were on anticoagulant therapy within five days of surgery, or had severe renal failure. Surgery was performed by a single surgeon using the modified Hardinge approach. The study drug was administered intracapsular after the capsule was closed, followed by the usual closure in layers. In addition to injecting the intracapsular study drug, the anesthesiologist also administered the intravenous study drug, as mentioned in Table .5 g of TEstimated blood loss was calculated using the difference between preoperative hemoglobin (Hb) and the final Hb before discharge, or, at the latest, day three (EBL). The formula by Good et al. was used to calculate the EBL . The amoA Hb of 80 g/L was considered to be an indication that blood products should be transfused.Measurements included transfusion rates, the average length of stay, 30-day readmissions, and complications. Deep vein thrombosis (DVT) with symptoms, pulmonary embolism (PE), and infection were considered to be complications.The sequences were obtained by a separate biostatistician. After that, pre-programmed group instructions for the placebo, intracapsular, and systemic groups were each given the sequence in a 1:1:1 ratio. These were then randomly split into three groups using computerized randomization.The chi-square test, one-way analysis of variance (ANOVA), followed by Tukey post hoc test, and paired t-test were used to analyze the variables.A total of 60 patients were recruited in our study. The demographic and clinical characteristics are presented in Table Preoperative mean Hb level was 133.4 ± 16.26 g/L in group A, 131.8 ± 14.83 g/L in group B, and 129.9 ± 14.85 g/L in group C. No significant difference was found in the mean Hb level among the groups (p = 0.771).At postoperative day three, the mean Hb level was 111.0 ± 13.70 g/L in group A, 111.5 ± 12.18 g/L in group B, and 100.20 ± 7.67 g/L in group C. A significant difference was found in the mean Hb level among the groups (p = 0.004). It was the maximum in group B and the minimum in group C , group B (p < 0.001), and group C (p < 0.001) Table .Comparison of Hb levels on day three between pairs of groups showed that there was a significant difference between group A and group C (p = 0.012) and between group B and group C (p = 0.008). However, no significant difference was found between group A and group B (p = 0.990) Table .Analysis of outcomes and complications of study groups is presented in Table Estimated blood loss was the maximum in group C and was found to be statistically significant when compared with other groups Table .The post hoc comparison of estimated blood loss between pairs of groups showed that there was a significant difference between group A and group C (p < 0.001) and between group B and group C (p < 0.001). However, no significant difference was found between group A and group C p = 0.712)  significantly lowers blood loss without increasing problems, which can eliminate concerns about intravenous TXA use. TXA lowers blood loss by 270 mL on average. We recommend the topical use of TXA in THA cases to decrease blood loss. Evidence also supports the use of TXA to reduce blood loss and the risk of blood transfusion after primary THA.


There have been very few real-world studies reported in the literature solely focusing on fremanezumab in Asia. This study aimed to evaluate the efficacy and safety of fremanezumab in a real-world setting in Japan.This single-centered, observational, retrospective study examined patients with migraines who received four doses of fremanezumab between December 2021 and August 2022 at Keio University Hospital. We assessed the changes in monthly migraine days, responder rates, and migraine-associated symptoms, as well as injection site reactions and adverse events.Twenty-nine patients were enrolled, wherein 79.3% were women. Compared with those at baseline, the monthly migraine days decreased by 5.9 days at 4 months. The 50% responder rate was 55.2% at 4 months. A total of 57.9%, 47.8%, and 65.0% of patients showed improvement in the severity of photophobia, phonophobia, and nausea/vomiting, respectively. Moreover, injection site reactions were the most common adverse events (55.2%).Fremanezumab is effective and safe for migraine prevention in Japan. Fremanezumab also improved migraine-associated symptoms in half of the patients.The online version contains supplementary material available at 10.1186/s12883-023-03449-3. Migraine is a neurological disorder with a high prevalence 8.4–14.4%) and burden on patients 4.4% and . MigrainTwo RW studies reported the efficacy and safety of fremanezumab in Italy , 15. TheWe conducted a single-center, observational, retrospective cohort study. This study was approved by the Ethics Committee of the Keio University School of Medicine , Tokyo, Japan. The patients were informed about this observational study via the institute’s website and could opt out of the study. The need for informed consent was waived by the Ethics Committee of the Keio University School of Medicine in accordance with national regulations  . The patPatients were administered fremanezumab 225 mg subcutaneously  at the first administration in our hospital. Next, the patients were administered 225 mg of subcutaneous fremanezumab monthly or 675 mg quarterly at the second visit, according to their preference. We started with a single dose of fremanezumab instead of three because each dose costs approximately 12,350 yen (88 USD as of June 2023) for most patients subscribing to the Japanese insurance system. During the study period, fremanezumab was only permitted in the syringe and not by self-injection in Japan; therefore, patients who did not prefer monthly visits to the hospital tended to choose quarterly dosing from the second dose.The inclusion criteria were as follows: treatment with fremanezumab for 4 months as the first CGRPmAb (de novo) either via monthly injections of fremanezumab 225 mg for four times or a dose of fremanezumab 225 mg at the first administration and a quarterly dose of fremanezumab 675 mg at the second administration from the headache group of the Keio University Hospital between December 2021  and August 2022; fulfillment of the diagnostic criteria for migraine, including probable migraine, according to the International Classification of Headache Disorders, 3rd edition (ICHD-3); and age ≥ 18 years. The patients were diagnosed with migraine by a headache specialist. Non-Asian patients were excluded  , 22 and Headache specialists explained the criteria for migraine based on the ICHD-3 to all patients, who were asked to track their headache and migraine days, including probable migraine days. Patients completed a questionnaire on monthly migraine days (MMD), monthly headache days (MHD), monthly days with acute medication use (AMD), pain intensity ), and associated symptoms  at baseline and after the first, second, third, and fourth months , severity, and other adverse reactions were also collected in the questionnaire. The patients were asked about their satisfaction levels at 4 months after receiving fremanezumab .We investigated the efficacy of the therapy by measuring the changes in MMD, MHD, AMD, NRS scores, and associated symptoms. The primary endpoints were a change in the MMD from baseline and 50% responder rate (RR) based on MMD. The secondary endpoints were changes from baseline in MHD; AMD; NRS; 25%, 75%, and 100% RR; and associated symptoms. We defined an improvement in associated symptoms as a reduction in symptom severity , and a disappearance as a disappearance of symptoms . For safety, we investigated the injection sites and reactions, and other adverse events .p < 0.05 [Data are presented as number (percent) and mean ± standard deviation. Differences from baseline in MMD, MHD, AMD, and NRS and their least-squares means were analyzed using the mixed-effects model for repeated measures, with time as a fixed effect and individual as a random effect. The correlation structure was defined as unstructured. Normality was assessed visually using residual plots. We did not impute missing data. The statistical analyses were performed using SAS version 9.4 . Statistical significance was set at p < 0.05 .n = 19; 225 mg/625 mg quarterly, n = 10)  or ineffectiveness, and 7 started fremanezumab later than 4 months before the end of the study period. We excluded one patient who switched from monthly to quarterly doses during the third administration. Twenty-nine patients were considered eligible for the efficacy and safety analyses  years. The mean MMD, MHD, and AMD were 12.6 ± 7.2 days/month, 14.7 ± 7.1 days/month, and 10.5 ± 7.3 days/month, respectively. At baseline, 34.5% and 24.1% of patients were diagnosed with CM and MOH, respectively , 3 (10.3%), 18 (62.1%), 7 (24.1%), and 4 (13.8%) patients had used lomerizine, propranolol, valproate, amitriptyline, and topiramate, respectively. Eleven (37.9%) patients used only one preventive drug, and the mean number of previous migraine preventives used was 1.9 ± 0.8. Twenty (69.0%) patients were using migraine prophylaxis at the time of initiating fremanezumab. Approximately half of the patients discontinued migraine prevention after the first dose of fremanezumab (Table p < 0.001) at 1 month, 6.0 days  at 2 months, 5.5 days  at 3 months, and 5.9 days  at 4 months. The MHD, AMD, and NRS scores were significantly reduced at 1 month compared to those at baseline  at 1 month, 4.4 days  at 2 months, 4.1 days  at 3 months, and 3.8 days  at 4 months  at 1 month, 8.6 days  at 2 months, 8.6 days  at 3 months, and 9.9 days  at 4 months  at 1 month, 9.7 days  at 2 months, 8.4 days  at 3 months, and 8.5 days  at 4 months   and 5 CM patients (50.0%), comprising 34.5% of studied patients, chose one dose of fremanezumab as the first dose and a quarterly dose of fremanezumab as the second dose. At baseline, the average MMD was 14.4 ± 8.6 days/month. Compared with this baseline, MMD decreased by 8.5 days , 52.6% , 57.9% , and 57.9%  of the patients at 1, 2, 3, and 4 months, respectively. An improvement in phonophobia was recorded in 56.5% , 50.0% , 54.5% , and 47.8%  of the patients at 1, 2, 3, and 4 months, respectively. An improvement in nausea/vomiting was recorded in 65.0% , 55.0% , 65.0% , and 65.0%  at 1, 2, 3, and 4 months, respectively  were very satisfied with the therapy, 10 (34.5%) were somewhat satisfied, and 4 (13.8%) were unsatisfied.To the best of our knowledge, this is the second RW study focusing solely on fremanezumab in Asian patients with migraine to be reported in an international journal . Our resThe efficacy of fremanezumab has been confirmed in randomized controlled trials, such as the HALO-EM and CM studies. In the HALO-EM study, the change in MMD was -3.7 days/month for monthly dosing and -3.4 days/month for quarterly dosing, and the 50% RR was 47.7% for monthly dosing and 44.4% for quarterly dosing . In the There was a multicenter RW cohort study on fremanezumab efficacy published in Italy , whereinan study . The 50%an study , 25. TheThere have been RW studies on CGRPmAbs, including fremanezumab, in Japan , 18. In In our study, 10 patients started with fremanezumab 225 mg as the first dose and changed to fremanezumab 675 mg (quarterly dosing) from the second month. These patients showed sustained benefit with a high 50% RR . In this RW study, it is not recommended to compare monthly vs quarterly dosing due to the possible selection bias. The dosing change was based on patients’ preferences. Those who preferred a quarterly dose in the beginning may have chosen to continue with the monthly dose if they were suspicious of the effect at 1 month, ending up in the monthly injection group.All three associated symptoms improved in the present study. In the Phase 3 HALO-EM study, fremanezumab reduced all three associated symptoms after 4 weeks. Fremanezumab significantly reduced the monthly average number of days with photophobia and phonophobia and nausea or vomiting from baseline for monthly  and quarterly  dosing strategies during the 12-week treatment period . EfficacAs for adverse reactions, injection site reactions were reported more frequently (up to 55.2% reported at least once) in our study compared to other real-world studies (< 9%) , 15, 19.No serious adverse events were observed in patients who received fremanezumab, and the most frequent adverse events were injection site reactions. However, three patients discontinued fremanezumab due to side effects . In our previous galcanezumab RW study, the most frequent injection reaction was pain, while in this study, the most frequent injection reaction was swelling . This diSatisfaction rate (very satisfied and somewhat satisfied) was high at 86.2%. High satisfaction rates with anti-CGRPmAbs have also been reported . ConsideThis study has several strengths. It is the second RW study from Asia that describes the efficacy and safety of fremanezumab solely in migraines. Moreover, to our knowledge, this is the first study to analyze the efficacy of changing the dosage from monthly to quarterly. Additionally, we analyzed the improvement in migraine-associated symptoms with fremanezumab, which has not been previously studied in the RW setting.However, this study had limitations including a small sample size, retrospective nature, single-center design, short observation period of 4 months, and selection bias regarding different schedules of administration. The primary endpoint (migraine days) was mainly assessed with questionnaires and not by the actual headache diaries, which were only checked in some cases , 20. ThuThis study revealed that fremanezumab is effective and safe for migraine prevention in Japan. Fremanezumab also improved migraine-associated symptoms in approximately half of the patients.Additional file 1: Supplementary Figure 1. Questionnaire.Additional file 2: Supplementary Figure 2. Responder rates at 1M for monthly and quarterly patients.


Voltammetric analysis of the electrochemical response of caffeine at the CF-µE clearly showed that no effects were attained due to the mass transport in solution. Differential pulse voltammetric analysis using the CF-µE was able to determine the detection sensitivity, concentration range (0.3 to 4.5 µmol L−1), limit of detection (0.13 μmol L−1) and linear relationship (I (µA) = (11.6 ± 0.09) × 10−3  − (0.37 ± 0.24) × 10−3), aiming at the quantification applicability in concentration quality-control for the beverages industry. When the homemade CF-µE was used to quantify the caffeine concentration in the soft beverage samples, the values obtained were satisfactory in comparison with the concentrations reported in the literature. Additionally, the concentrations were analytically determined by high-performance liquid chromatography (HPLC). These results show that these electrodes may be an alternative to the development of new and portable reliable analytical tools at low cost with high efficiency.In the development of electrochemical sensors, carbon micro-structured or micro-materials have been widely used as supports/modifiers to improve the performance of bare electrodes. In the case of carbon fibers (CFs), these carbonaceous materials have received extensive attention and their use has been proposed in a variety of fields. However, to the best of our knowledge, no attempts for electroanalytical determination of caffeine with CF microelectrode (µE) have been reported in the literature. Therefore, a homemade CF-µE was fabricated, characterized, and used to determine caffeine in soft beverage samples. From the electrochemical characterization of the CF-µE in K Caffeine or 1-3-7-trimethylxantine is a white crystalline xanthine alkaloid that promotes various effects on the body’s metabolism when ingested, including stimulating the central nervous system, increasing blood pressure in the short term, and secreting gastric acid. This drug is widely used in different concentrations in coffee, cola nuts, cocoa beans, tea leaves, cola beverages and several pharmaceutical substances used worldwide ,3. ThereThe most common technique used for analyzing/quantifying this bioactive compound is high-performance liquid chromatography (HPLC) ,4,5,6,7.Concerning the electroanalytical detection of caffeine, various sensing-materials have been examined and reported in the existing literature; however, these electrode materials are expensive, and their preparation methodologies are dependent on time-consuming procedures and modification steps sensor ,21,22,23In the development of electrochemical microsensors, carbon materials have been widely used as supports due to their simplicity and active surface to improve the performances in specific concentration control areas ,31. AmonBased on the existing literature, only few reports have described the applicability of carbon-based sensors to determine caffeine in food, beverages, drugs, and medications. However, to the best of our knowledge, no attempts for electroanalytical determination of caffeine with CF-µE have been reported in the literature. Therefore, a homemade CF-µE was fabricated, characterized, and used to determine caffeine, estimating the concentration linear range, calibration function, and determination of caffeine in soft beverage samples.2SO4 and caffeine (purity 98%) were purchased from Quimex and Isofar, respectively. K₃[Fe(CN)₆] and KCl were purchased from Synth . Solutions were daily prepared under constant agitation for 30 min before each experiment.All reagents were of analytical grade, and all aqueous solutions were prepared using a high-purity water obtained from a Millipore Milli-Q system with resistivity >18 MΩ at 25 °C. HA cleaned 3 cm length carbon fiber was connected to a Cu wire (⌀ 1 mm diameter and 12 cm length) with silver conductive ink  Brazil). After drying, this set was carefully inserted into a plastic mold, filled with epoxy resin SQ 2119-PT (Avipol (SP), Brazil), and held in an upright position. For curing and demolding, the 48-hour time was respected. Then, the CF-µE was polished using 1200 and 1500 grit sandpaper until obtaining a smooth and flat surface containing a carbon fiber microdisk. Finally, the microelectrode was washed thoroughly with distilled water, air-dried, inspected by using an optical microscope  to prevent any defects and stored away from dust. A scheme for the CF-µE fabrication is provided in the −1 KCl) as a reference electrode, a platinum wire as a counter electrode, and a homemade CF-µE as the working electrode. The CF-µE radius was estimated recording a CV in K3Fe(CN)6 10 mmol L−1 plus KCl 100 mmol L−1 solution. The caffeine electroanalytical experiments were carried out at 25 °C and five CVs were recorded for each measurement in the potential range from +0.5 to +1.7 V with scan rate of 50 mV s−1 in 0.5 mol L−1 H2SO4 as the supporting electrolyte in the presence and absence of caffeine 0.1 mol L−1. The effect of the scan rate was also studied at 20, 40, 60, 80, and 100 mV s−1 in the presence of caffeine in 0.5 mol L−1 H2SO4. The DPV parameters to quantify caffeine, using 0.5 mol L−1 H2SO4 as a supporting electrolyte, were equilibration time = 10 s, initial potential = +0.5 V, final potential = +1.7 V, potential scan rate = 50 mV s−1, pulse amplitude = +0.05 V, and modulation time: 0.04 s. The above-optimized parameters were used for all measurements. Then, the calibrations curves (peak intensity vs. caffeine concentration in the range from 0 to 6 µmol L−1) were studied by least-square linear regression, and the obtained figures (slopes and intercepts) were reported with their confidence interval, p = 95%. Reproducibility and stability parameters were also evaluated. Homemade CF-µE was cleaned recording ten CV cycles from +0.60 V to +1.80 V at 100 mV s−1 in 0.5 mol L−1 H2SO4 before each one of the measurements. Caffeine determinations with CF-µE were validated by a reverse-phase HPLC  equipped with a Nucleosil C18 column, Berlin, Germany (4.6 × 250 mm), and an UV–vis detector set at 273 nm. An acetonitrile/water mixture (25:75 % v/v) was used as the mobile phase at a flow rate of 0.6 mL min−1, injecting 20 μL of each sample. The retention time (tr) was 6.8 min.Voltammetric analysis (cyclic voltammetry (CV) and differential pulse voltammetry (DPV)) were performed with an Autolab PGSTAT302 by using a three-electrode cell consisting of an Ag/AgCl  was used to eliminate the gas from commercial soft drinks, which were then transferred to the electrochemical cell with the supporting electrolyte to proceed with the caffeine detection (0.5 mL to 4.5 mL of supporting electrolyte). The standard addition method was used to quantify caffeine in the samples to minimize the possible matrix effects due to the presence of other components in the real samples. All experiments were carried out in triplicate, and mean values (standard deviation < 5%) were used for the figures.3Fe(CN)6 10 mmol L−1 plus KCl 100 mmol L−1 solution. In IL is the limiting current at the steady-state condition (A), n is the number of electrons involved on the electrodic reaction, F is the Faraday constant , D is the diffusion coefficient (cm2 s−1), C is the bulk concentration of the electroactive species (mol cm−3), and r is the radius of the microdisk electrode (cm)  – (0.37 ± 0.24) × 10−3 . The limit of detection (LOD) was estimated from the equation LOD = 3.3 × Sy/x/b, where y/xS is the residual standard deviation and b is the slope of the calibration plot, which was approximately 0.13 μmol L−1. Additionally, the residuals of the regression were randomly distributed around the zero  were conducted in the electrochemical cell ,55. FiguThe mean results were obtained for the standard additions protocol at each one of the soft drinks analyzed, recording three measurements with acceptable standard deviations and confidence intervals relating to a probability of 95%. This strategy consents to validate both false positives and false negatives (α = β = 0.05), as recommended by the IUPAC ,39. SubsAs described, CF-µE presented good performance for quantifying caffeine. The selectivity of this sensor was also evaluated by intentionally introducing concentrations of ascorbic acid as interference during caffeine analysis. Ascorbic acid, also known as Vitamin C, is a water-soluble vitamin found in citrus and other fruits and vegetables; also, it is a supplement in the soft beverages. Therefore, this acid was chosen to test the determination of caffeine in the presence of an important interference. The experimental data are reported in A homemade CF-µE was fabricated and it was a suitable electrochemical microsensor for caffeine determinations. Under the experimental conditions examined, caffeine displayed irreversible behavior in cyclic voltammetry with the CF-µE. This sensor exhibited a high detection sensitivity, a high mass transfer rate, and an enhanced signal-to-noise ratio; consequently, it is shown to have extraordinary stability and reproducibility, making it more applicable as a sensor in the food control analyses and measurements. For future works of this research field, some modifiers could be investigated in order to increase the surface area of the microsensor to guarantee more stability, sensibility, and selectivity for real-time monitoring.


Alkalihalobacillus clausii isolate, three Bacillus safensis isolates, five Escherichia coli isolates, and one Pasteurella multocida isolate.Here, we present the first draft genome sequences of 10 bacterial strains that were isolated from the rumen, nasopharynx, vagina, or uterus of healthy beef cattle. These genomes are from one  Ten bacterial strains were isolated from ruminal fluid and deep nasopharyngeal, vaginal, and uterine swab samples collected from Angus-based steers, pregnant and nonpregnant heifers, and cows born and raised at the Central Grasslands Research Extension Center . Samples were collected from the nasopharynx, rumen, and vagina as described previously . The ute2. For genomic DNA extraction, each cryopreserved isolate was regrown in brain heart infusion broth (BD) overnight at 37°C with 5% CO2, and 1 mL of this culture was centrifuged at 8,000 × g for 10 min at 4°C. DNA was then extracted from the cell pellet using the DNeasy blood and tissue kit , with modifications as detailed in our previous publication  and incubating the plates overnight at 37°C with 5% COhttps://sourceforge.net/projects/bbmap) with the minlength parameter set to 500 bp. Completeness and contamination were calculated with CheckM v.1.2.0  and GenBank, respectively, under the accession numbers listed in


C. weissflogii are unclear. In this study, single-factor experiments are designed and followed by an orthogonal experiment to determine the optimal combination of fucoxanthin production conditions in C. weissflogii. The results showed that the optimum conditions for fucoxanthin production were a temperature of 30 °C, a light intensity of 30 umol m−2 s−1 and a salinity of 25. Under these conditions, the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii reached 1.97 × 106 cell mL−1, 0.76 g L−1, 2.209 mg L−1 and 1.372 mg g−1, respectively, which were increased to 1.53, 1.71, 2.50 and 1.48 times higher than their initial content. The work sought to give useful information that will lead to an improved understanding of the effective method of cultivation of C. weissflogii for natural fucoxanthin production.Fucoxanthin is a natural active substance derived from diatoms that is beneficial to the growth and immunity of humans and aquatic animals. Temperature, light and salinity are important environmental factors affecting the accumulation of diatom actives; however, their effects on the production of fucoxanthin in  The new health paradigm towards disease prevention has led to an increasing focus on nutraceuticals with both nutritional and pharmaceutical benefits. Nutraceuticals made from natural derivatives are becoming more and more popular due to their safety. Natural carotenoids, naturally synthesized by algae, plants, yeast and bacteria, are rapidly emerging in nutraceuticals and functional foods . FucoxanPresently, the main source of commercialization of fucoxanthin is brown seaweeds . HoweverConticribra weissflogii is a typical marine planktonic diatom which has the characteristics of fast growth, strong adaptability, being rich in polyunsaturated fatty acids, etc., and is often used as live feed by aquaculturists for large-scale cultivation [C. weissflogii is competitive as a potential fucoxanthin producer, driven by the success of its large-scale cultivation. However, the optimization of abiotic parameters for fucoxanthin production from C. weissflogii remain a big challenge. In this work, the effects of temperatures, light intensities and salinities on the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii were studied, and an attempt was made to determine the optimal combination for the abiotic parameters of fucoxanthin production via orthogonal experiments. These results are valuable for optimizing the abiotic parameters of fucoxanthin production by C. weissflogii.Marine diatoms are rich in fucoxanthin, which can contain up to 100 times more than brown seaweeds , and aretivation ,9. C. weC. weissflogii . Similarly, the fucoxanthin content in the 30 °C group was significantly higher, with a maximum of 1.36 ± 0.02 mg g−1. The results showed that different temperatures had significant effects on the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii.From −1 d−1 for the 10-day cultivation periods .The results a showed −2 s−1 group, which showed continuous growth. The maximum value appeared on the 10th day of 30 μmol m−2 s−1. At this time, the content of carotenoids in C. weissflogii was 1.24 ± 0.03 mg L−1, which was 1.76 times higher than the initial content (0.45 mg L−1). According to −2 s−1 group, the fucoxanthin content of the other experimental groups increased first and then decreased, and the maximum value appeared on the 8th day. At this time, the fucoxanthin content of the 30 μmol m−2 s−1 experimental group was 0.99 ± 0.01 mg g−1, which was 1.27 times higher than the initial content (0.44 ± 0.01 mg g −1). It was proved that different light intensities had significant effects on the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii.It can be seen from C. weissflogii experienced maximum fucoxanthin productivity under 0, 60, 90 and 120 μmol m−2 s−1 after day 4 of the cultivation period, while under 30 μmol m−2 s−1, the maximum productivity of fucoxanthin was obtained until the 8th day of cultivation  with a maximum value of 0.07 mg g−1 d−1. Therefore, 30 μmol m−2 s−1 could serve as the optimum light intensity for the maximum production of fucoxanthin.  compared with other experimental groups. A similar trend was observed in biomass production (−1) was obtained in the 25‰ group, which was significantly (p < 0.05) higher than the other experimental groups.As shown in oduction b. The maC. weissflogii compared with the other experimental groups with 1.24 ± 0.03 mg L−1, which was 1.76 times higher than the initial value of 0.45 ± 0.04 mg L−1. According to C. weissflogii was observed in the 25‰ group. The maximum value of fucoxanthin content was reached, 0.99 ± 0.01 mg g−1, on the 8th day of cultivation, which was 1.25 times higher than the initial content (0.44 ± 0.01 mg g−1). The results demonstrated that different salinities had significant effects on the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii.It can be observed from −1 d−1  orthogonal test  intensity [Haematococcus pluvialis is suitable for growth in low-light conditions (<100 μmol m−2 s−1), while astaxanthin requires strong light conditions (>200 μmol m−2 s−1) to achieve more efficient accumulation [−2 s−1 [C. weissflogii was cultured in a light intensity of 0–120 μmol m−2 s−1. The results showed that when the light intensity was 30 μmol m−2 s−1, the algal cell density, biomass, carotenoid content and fucoxanthin content reached the maximum. This also confirmed the findings of Li et al. [C. weissflogii is a kind of microalgae that is easy to culture under low-light conditions.Marine microalgae are mainly autotrophic through light energy, and the efficiency of photosynthesis plays a vital role in the growth of algae. Therefore, light intensity could directly affect the growth rate, photosynthetic activities and biochemical components of microalgae ,19,20. S m−2 s−1 . The opt m−2 s−1 . The algntensity . Howevermulation . Since f [−2 s−1 . In thisi et al. . C. weisIsochrysis galbana exhibited a higher growth and fucoxanthin content than 20‰ salinity at 35‰ salinity [Phaeodactylum tricornutum and fucoxanthin content was optimal at salinity levels of 20‰ [Cylindrotheca fusiformis, was observed to grow best at a salinity of 30‰, while fucoxanthin content was the highest at a salinity of 10‰ [Salinity can affect the osmotic pressure, nutrient absorption and suspension of algae to a certain extent. When the salinity of the aquaculture water where the microalgae cells are located changes, the osmotic pressure of the cells will also change . The micsalinity . An optis of 20‰ . The othy of 10‰ . In thisC. weissflogi was isolated and purified from a shrimp pond in southern China in 2019 by the Laboratory of Algal Resources Development and Ecological Remediation of Aquaculture Environment, Guangdong Ocean University, Zhanjiang, China. Stock cultures of C. weissflogi were maintained at 25 ± 1 °C under a continuous light intensity of 30 μmol m−2 s−1 in 1 L flasks. A modified version of F/2 media was used, and the detailed formulation can be found in our recent study [The microalgae nt study . We cent−2 s−1) and salinity . Temperature single-factor experiments were performed at 30 μmol m−2 s−1 and 25‰. Light single-factor experiments were performed at 25 °C and 25‰. Salinity single-factor experiments were performed at 25 °C and 30 μmol m−2 s−1. The culture volume for all cultures was 700 mL, and the initial algae density was about 6 × 105 cell mL−1. During the experiment, all cultures were continuously illuminated, sealed and not inflated. Three parallels were set for each treatment, and the experiment period was 10 days.Five concentration gradients were set up for each experiment group in single-factor experiments of temperature , light intensity  under a BX53 fluorescence microscope after being fixed with Lugol’s iodine solution [The cell density of solution .1). Wash twice with distilled water in this process. The membrane containing algal cells is then placed in an oven and dried at 90 °C for 500 min to constant weight. The total mass M2 is measured and recorded. The biomass content was calculated according to the following formula.Biomass was determined using the drying differential weight method . Filter −1 for 10 min, discard the supernatant, add 10 mL of ethanol with a volume fraction of 95%, dark-treat 24 h, centrifuge at 5000 r min−1 for 10 min and measure the supernatant with a spectrophotometer The optical density value D of the solution was measured at 480, 510 and 750 nm. The content of carotenoids was calculated according to the following formula.Using ethanol extraction method , take 10−1, 10 min) and remove the supernatant. Add absolute ethanol to make a material-to-liquid ratio of 1 g:40 mL, and then extract twice for 1 h each while protected from light at 60 °C. After leaching, the supernatant is collected via centrifugation  and the absorbance (D445) is determined with a UV spectrophotometer at 445 nm. The fucoxanthin content C can be expressed as:Extraction with organic solvents . Take 80In the formula, N is the dilution ratio; V is the volume of the crude extract; A′ is the theoretical absorption value of a solute with a volume fraction of 1% in a colorimetric cup of 1 cm optical range length and has a value of 1600; and M is the sample mass.p < 0.05 indicated that there was a significant difference.Excel 2019 was used to process the data and draw charts. SPSS 26 was used to analyze the single-factor analysis of variance and orthogonal range analysis of each index to test the differences between the data of each group. C. weissflogii. Settings of 30 °C, 30 μmol m−2 s−1 and 25‰ were determined to be the optimum condition for biomass and fucoxanthin production. After 8 days of cultivation under these conditions, the cell density, biomass, carotenoid content and fucoxanthin content of C. weissflogii reached 1.97 × 106 cell mL−1, 0.76 g L−1, 2.209 mg L−1 and 1.372 mg g−1, respectively, which were increased to 1.53, 1.71, 2.50 and 1.48 times higher than their initial content. Cultivated environmental factor optimization improved biomass and fucoxanthin production, but the efficient production of fucoxanthin by C. weissflogii still required a lot of work to do..In this study, the single-factor experiment and orthogonal experiment were used to optimize the biomass and fucoxanthin production conditions of