Thymus daenensis, Nepeta sessilifolia, Hymenocrater incanus, and Stachys inflata). In particular, only the composition of essential oils from H. incanus was completely detected (99.13%), while that of the others was only partially detected. Oxygenated monoterpenes (75.57%) were the main compounds of essential oil from T. daenensis; sesquiterpenes hydrocarbons (26.88%) were the most abundant in S. inflata; oxygenated sesquiterpenes (41.22%) were mainly detected in H. incanus essential oil, while the essential oil from N. sessilifolia was mainly composed of non-terpene and fatty acids (77.18%). Due to their slightly different composition, also the antibacterial activity was affected by the essential oil tested. Indeed, the highest antibacterial and antifungal activities were obtained with the essential oil from T. daenensis by means of the inhibition halo (39 ± 1 and 25 ± 0 mm) against Gram-positive strains such as Staphylococcus aureus and Aspergillus brasiliensis. The minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC) of the essential oils obtained from the four species varied from 16 to 2000 μg/mL and were strictly affected by the type of microorganism tested. As an example, the essential oils from H. incanus and S. inflata were the most effective against the Gram-negative bacterium Pseudomonas aeruginosa , which is considered one of the most resistant bacterial strain. Therefore, the essential oils obtained from the four species contained a suitable phytocomplexes with potential applications in different commercial area such as agriculture, food, pharmaceutical and cosmetic industries. Moreover, these essential oils can be considered a valuable natural alternative to some synthetic antibiotics, thanks to their ability to control the growth of different bacteria and fungi.In this study the essential oils obtained from four different plant species belonging to the Lamiaceae family were extracted by means of hydrodistillation and their composition and antimicrobial activity were evaluated. About 66 components were identified by using gas chromatography-mass spectrometry (GC–MS), and among all, thymol (67.7%), oleic acid (0.5–62.1%), (−)-caryophyllene oxide (0.4–24.8%), α-pinene (1.1–19.4%), 1,8-cineole (0.2–15.4%), palmitic acid (0.32–13.28%), ( +)spathulenol (11.16%), and germacrene D (0.3–10.3%) were the most abundant in all the species tested (i.e.  To this purpose the scientific community has focused its attention on natural, safe and effective antimicrobial molecules. In particular, essential oils have been traditionally used for their antimicrobial effects as topical or systemic drugs for bacterial and fungal infections, as preservative in food and topical ointments, as natural as biocide in ecological agriculture. The different molecules contained in the essential oils can exsert a synergistic effect provide a protection higher than that achieved by single molecules also reducing the multidrug resistance, which occurs in different infections and and inhibiting the contamination by foodborne pathogens5.An increased interest in finding new and safe antimicrobial molecules from natural origin, especially from plants, has been detected in the last decades6. The antifungal and antimicrobial effect of several essential oils from this family, like thyme, peppermint, lavender, rosemary, peppermint, savory and marjoram, has been previous demonstrated8. The essential oil obtained from Thymus daenensis Čelak., endemic in NE Iraq and Iran, contains thymol, carvacrol, and p-cymene in high amount15. Thymol and β-caryophyllene, being the main components of T. daenensis essential oil, seem to be responsible of its antifungal and antibacterial effects19. As a function of its composition and origin, the essential oils of T. daenensis have been used to treat asthma, recurrent dry cough, and bronchitis as well as food ingredient20. The antimicrobial activity of some Nepeta species has been studied as well23. The essential oil from Nepeta sessilifolia Bunge, endemic in Iran and Pakistan, was especially effective against Candida albicans. β-caryophyllene and 1,8-cineole, α-pinene, β-pinene, trans-β-ocimene, germacrene-D, and caryophyllene oxide are the major constituents of the essential oil of Hymenocrater incanus Bunge an exclusive species of this genus in Iran24. Recent studies confirmed the potential of the secondary metabolites from this genus as antimicrobial antifungal, antiparasitic27. Germacrene-D, bicyclogermacrene, and α-pinene are the main components contained in the essential of Stachys inflata Benth., native from NE Turkey to Iran, which have antimicrobial effect, especially against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa32.Essential oils obtained from Lamiaceae have gained considerable interest since their rich content in biological active molecules especially volatile molecules, such as monoterpenes, sesquiterpenes, and coumarins in some casesBacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella ser. Enteritidis, Salmonella ser. Typhimurium and Staphylococcus aureus.The recent interest of consumers in food preservatives of natural origin has shifted the research interest towards the possible application of the essential oils to this propose. Several in vitro studies have established the efficacy of essential oils from Lamiaceae family taxa against common foodborne pathogens such as Overall results disclosed that the cultivation region can strongly affect the composition and consequently the effectiveness of essential oils from Lamiaceae and their selectivity against different pathogens. Given that, it is important to simultaneously compare the efficacy of essential oils obtained from different species also testing different pathogens.Accordingly, in this study, the essential oils from four different species of Lamiaceae were extracted and characterized and their efficacy was tested simultaneously using 12 strains of microorganisms.T. daenensis, N. sessilifolia, H. incanus, and S. inflata, were collected from the Daran region, located in Isfahan province of Iran (longitude: 46˚49ʹ02ʺ and latitude: 36˚54ʹ170ʺ). Permission for collection of plant materials obtained from the Agricultural Jahad Office and also the owner of the farm. The study is in compliance with relevant institutional, national, and international guidelines and legislation. The harvested specimens were transferred to the laboratory and exposed to free air in shade to dry. One sample of each whole plant was collected and pressed. The plant was identified and recorded at the herbarium of the University of Kashan. The plant was identified and recorded with code number 1018, 1019, 1020, 1021.Aerial parts of 60.After complete drying, the samples were grinded to obtain small particles and ensure the complete extraction of the bioactives; 100 g of each sample was subjected to extraction by means of hydrodistillation using a Clevenger apparatus for 5 h. The weight of essential oils collected after sodium sulphate dehydration was calculated accurately and essential oils was stored in closed bottles at 4 °C in the dark until further use33.The determination of the constituents of essential oil samples has been performed by means of GC–MS method. A chromatograph (Model 6890) Coupled with an N-5973 mass spectrometer made in USA and a HP-5MS Capillary Column with 5% methylphenylsiloxane static phase  and ionization energy of 70 eV has been used for qualitative identification of the components. A temperature gradient has been used for the analysis, starting from 60 °C and then increasing the temperature (at a rate of 3 °C/min) up to 246 °C. The injector and detector temperature were set at 250 °C, the injection volume was 1 µl with 1.50 split and the helium carrier gas delivered at a flow rate of 1.5 ml/min. The chemical components of the essential oils were identified as a function of the retention indices about standards of n-alkane mixtures (C8–C20) and mass spectral data of each peak using a computer library  and comparing these data with those reported in the literatureStaphylococcus epidermidis (ATCC 12228), S. aureus (ATCC 29737) and Bacillus subtilis (ATCC 6633) were chosen as Gram-positive bacteria, while Klebsiella pneumonia (ATCC 10031), Shigella dysenteriae (PTCC 1188), Pseudomonas aeruginosa (ATCC 27853), Salmonella paratyphi-A serotype (ATCC 5702), Proteus vulgaris (PTCC 1182) and Escherichia coli (ATCC 10536) were the Gram-negative bacteria selected. Aspergillus niger (ATCC 16404), Aspergillus brasiliensis (PTCC 5011) and Candida albicans (ATCC 10231) were the fungal strains tested.Twelve microorganisms were used to evaluate the antimicrobial activity of the selected essential oils. Microbial strains were provided by the Iranian Research Organization for Science and Technology . 61.Well plates 6.0 mm in diameter containing Müller Hinton agar were prepared, and 100 µL of bacterial suspensions with a half-McFarland turbidity equivalent in culture medium were cultured. The essential oil (30 mg/mL) was dissolved in dimethylsulfoxide (DMSO) and 10 μL  of each essential oil was poured into the wells. The plates were incubated at 37 °C for 24 h and their antimicrobial activity was measured for each microorganism measuring, by the antibiogram ruler (in millimetres), the diameter of inhibition halos. Three replicates were performed for each strain and results were calculated as mean ± standard deviation. DMSO was used as negative control and gentamicin and rifampin for bacteria, and nystatin for fungi, were used to compare their antimicrobial power with those of the essential oils61. The MIC was determined by the improvement of opacity or the change in colour as the lowest concentration that inhibited the visible growth (absence of turbidity).The minimum concentration capable of inhibiting the growth of the bacteria or fungi (MIC) was calculated by microdilution method. The essential oils were dissolved in a mixture of TSB medium and DMSO at an initial concentration of 2000 μg/mL. The stock solutions were diluted to reach the following concentrations: 1000, 500, 250, 125, 62.5, 31.25 and 15.63 µg/mL. Experiments were performed by using 96-well microplates. 95 µL of culture medium, 5 µl of bacterial suspension with 0.5 McFarland dilution, and 100 µL of essential oil dilutions were added to each well, and then the plate was incubated at 37 °C for 24 h for bacterial strains and 48 h and 72 h at 30 °C for yeast61. The MBC/MFC was the lowest concentration able to effectively reduce the growth of microorganisms (99.5%).To determine the minimum concentration capable of killing the bacterial or fungal strains, the same microdilution method was used. After 24 h of incubation of bacteria with the essential oils at different concentrations, 5 µL of the content of each well were inoculated with neutrin agar medium and incubated at 37 °C for 24 h for bacterial strains and 48 h and 72 h at 30 °C for yeasts. After incubation, the colony-forming units (CFUs) were enumeratedt-test were performed to substantiate differences between groups using XL Statistics for Windows. The differences were considered statistically significant for p < 0.05.Results are expressed as the mean ± standard deviation. Analysis of variance (ANOVA) was used for multiple comparisons of means, and the Tukey’s test and Student’s T. daenensis, 0.2% from N. sessilifolia, 0.02% from H. incanus, and 0.14% from S. inflata.The different essential oils were obtained from the four different Lamiaceae species from Iran. Their colour varied from pale yellow to dark yellow and the yield was 1.88% from H. incanus was completely detected (99.13%), while that of T. daenensis (97.44%), S. inflata (95.77%), and N. sessilifolia (84.56%), was only partially detected (Table T. daenensis were oxygenated monoterpenes (75.57%), from S. inflata were sesquiterpenes hydrocarbons (26.88%) and from H. incanus were oxygenated sesquiterpenes (41.22%). Differently, the essential oil from N. sessilifolia was mainly composed of non-terpene and fatty acids (77.18%).The chemical composition of the essential oils was investigated using a GC–MS. About 51 components were identified. The composition of essential oils from T. daenensis was also rich in monoterpenes hydrocarbons (18.3%), and its main components were thymol (67.71%), γ-terpinene (6.20%), p-cymene (5.16%) and borneol (3.67%), and 1,8-cineole (15%)  and oxygenated sesquiterpenes (41.22%). In particular, (−)-caryophyllene oxide (24.81%), oleic acid (23.53%), palmitic acid (28.23%), phytol (9.22%), α-cadinol (7.66%), and caryophyllenol-II were found (5.06%) , palmitic acid (12.12%), ( +)spathulenol (11.16%), germacrene D (12.26%), and bicyclogermacrene (9.9%) were detected (Table Acids 42.22%) and sesquiterpene hydrocarbons (26.25%), were the major constituents of the essential oil from tic acid 2.12%,  and the lowest in essential oil from H. incanus (0.22%). The highest amount of oleic acid was detected in the oil from N. sessilifolia (62.09%) while the lowest in that from T. daenensis (0.49%). Linalool was found in all essential oils except in that from H. incanus. Trans-Caryophyllene and (−)-Caryophyllene oxide were found in all essential oils except in that from S. inflata and the highest amount (3.68% and 24.81%) was found in oil from H. incanus. Thymol (67.71) and p-cymene (5.16) were contained only in the oil from T. daenensis; linoleic acid (6.06) and S. inflata; caryophyllenol-II (5.06) in the oil from H. incanus, ( +) spathulenol (11.16) and in the oil from S. inflata.Overall data underlined that 1,8-cineole and oleic acid were commonly present in the essential oils of S. aureus with essential oil from T. daenensis, and it was even higher than that obtained by using rifampin (21 ± 0 mm) and gentamicin (27 ± 0 mm). Essential oil from N. sessilifolia (10 ± 1 mm) and S. inflata (9 ± 1 mm) had a significantly lower activity against this bacterium, while oil from H. incanus did not show any activity.The antibacterial and antifungal activities of the essential oils were measured to evaluate their potential applications Table . The higT. daenensis was also effective against A. brasiliensis (25 ± 0 mm), and its activity was similar to that of nystatin (30 ± 0 mm), used as control. Moreover, oil from T. daenensis was capable of inhibiting the growth of A. sniger (12 ± 0 mm) and C. albicans (12 ± 1 mm) but in a lesser extent than nystatin (27 ± 0 and 33 ± 0 mm).Essential oil from T. daenensis was also effective in counteracting two Gram-negative bacteria K. pneumonia (18 ± 1 mm) mm and Sh. dysenteriae (16 ± 0 mm) as the inhibition halos were larger than those obtained treating the same bacteria with rifampin (8 ± 0 mm and 9 ± 0 mm) and gentamicin (17 ± 0 mm). The oil was also effective against two more Gram-negative bacteria E. coli and S. paratyphi-A, being the inhibition halo 11 ± 1 and 12 ± 1 mm, which was slightly larger than that obtained by using rifampin (10 ± 0 and 8 ± 0 mm) and lower than that exerted by gentamicin (23 ± 0 and 18 ± 0 mm). It was also slightly effective against the Gram-positive S. epidermidis (9 ± 0), but any effect was detected against S. paratyphi-A, S. epidermidis, and P. aeruginosa.Essential oil from N. sessilifolia was less effective than the essential oil obtained from T. daenensis as it inhibited the growth of only three Gram-positive bacteria: B. subtilis (14 ± 1 mm), S. epidermidis (9 ± 0 mm), and S. aureus (10 ± 1 mm). The efficacy of this oil was also lower than that obtained by using rifampin and gentamicin.Essential oil from H. incanus inhibited only B. subtilis (10 ± 0) and S. epidermidis (11 ± 0 mm) in the less extend than rifampin and gentamicin. Essential oil from S. inflata had a weak activity against Gram-positive bacteria like B. subtilis (9 ± 0 mm) and S. aureus (9 ± 1 mm).Essential oil from P. aeruginosa with essential oil from S. inflata were lower than that obtained using rifampin (31 μg/mL), but it was significantly higher than that obtained using gentamicin (8 µg/mL). The other essential oils had higher MIC (125 µg/mL) against this bacterium. The MIC of all the used oils (except that from T. daenensis) against A. brasiliensis and A. niger were around 2000 µg/mL, disclosing their inactivity and essential oil from S. inflata was not effective also against S. aureus (MIC > 1000 μg/mL).The MIC of the essential oils varied from > 16 μg/mL to > 2000 μg/mL as a function of the microorganism and oil used Table . The MICT. daenensis varied between 125 and 500 μg/mL, with its weakest inhibitory effect against Sh. dysenteriae. The antifungal activity of oil from T. daenensis was slightly higher (MIC from 31 to 250 μg/mL) and it especially inhibited C. albicans as its power was three times higher than that of nystatin (125 μg/mL).The MIC obtained treating the different bacteria with the oil from T. daenensis varied from 63 to 500 µg/mL . The lowest MBC of the essential oil from N. sessilifolia was obtained against Sh. dysenteriae and K. pneumonia, confirming its ability to inhibit and kill them. The low efficacy and high MICs and MBCs of essential oil from N. sessilifolia against most of the bacterial strains tested in this work, should be connected with the absence of terpenes (which are considered the most effective against bacteria). The antimicrobial power against few bacterial strains should be related to the fatty acid content, even if the mechanism of action of these compounds is still completely unknown and they are supposed to modify the permeability of the membrane and promote its disruption, thus causing significant alterations on the membrane-dependent conduction systems.The MICs obtained by treating all microorganisms with essential oil from H. incanus varied between > 16 and > 2000 µg/mL. The strongest activity was found against the Gram-negative P. aeruginosa (MIC 63 μg/mL). The MIC of this oil against C. albicans was (63 µg/mL) lower than that of nystatin (MIC 125 µg/mL) while the MFC was significantly higher (250 µg/mL). Treating the other microorganisms, MBCs/MFCs were always equal to MICs and oxygenated sesquiterpenes such as (−)-caryophyllene oxide and α-cadinol contained can be responsible of these activities.The MIC values obtained treating the different microorganisms with oil from S. inflata against the tested microorganisms varied between > 16 and > 2000 μg/mL. The strongest effect was found against the Gram-negative P. aeruginosa, being the MIC very low (16 μg/mL).The MICs essential oil from T. daenensis was 2.09%15, from N. sessilifolia was 0.56%34, from H. incanus was 0.6%24, and from S. inflata was 2.9%31. Then the yields were always higher probably because the used plants were grown in different areas where the environmental factors can strongly affect the content of secondary metabolites35.In previous studies, the yield of essential oil collected from T. daenensis from the Kurdistan region of Iran14. Due to the high content of thymol, T. daenensis can be considered as the main source of this valuable compound, which is the phenolic compounds with remarkable antimicrobial properties38.The obtained results were in line with those reported previously, as thymol was always the main component, and the highest amount (91.15%) has been detected with 40, reported that oxygenated sesquiterpene (35.3% and 33.14%) and oxygenated monoterpene (49%) were the main components of this essential oil obtained from plants collected in Ghamshelo and Arak, Iran. Hence, the high content of acids is a unique characteristic of the plants harvested from Isfahan province of Iran and can be strongly affected by the location, climatic and ecological conditions, field operations, growth stage, and genetic traits41. Thanks to its acid content, the essential oil from N. sessilifolia can exert several beneficial activities. Indeed, oleic Acid (9-Octadecenoic acid) is a component of omega-9 fatty acids, capable of counteracting cancer and cardiovascular diseases, autoimmune diseases, Parkinson’s and Alzheimer’s diseases, inflammatory diseases and hypertension44. Linoleic acid is one of the most unsaturated fatty acids of the human diet except for omega-6 fatty acids and has an active role in human growth and general health42.Differently,24 because only some compounds were similar but the most were different and none of the major constituents found in this essential oil was recorded by24. These results confirmed that environmental and climate conditions have a significant impact on the chemotypic properties of the obtained oil45. Caryophyllene oxide, which is contained in high amount in the oil from H. incanus collected in the Isfahan province of Iran, can inhibit the abnormal accumulation of fluid in the intercellular space of body tissues and it has been used as antitumor agent46.This result is not completely in agreement with those ofS. inflata, while palmitic acid (9.1%) has been indicated as the most abundant bioactive of the essential oil obtained from this plant by28. Germacrene D and bicyclogermacrene were found by other authors but in different amount: 8.9% and 5.1%28, 16.9% and 16.6%29, and 32.9% and 7.3%30. The differences found were mainly related to genetic or non-genetic variations connected with environmental differences such as soil chemical composition and physiographic factors.In none of the previous studies, oleic acid has been reported as main component of the essential oil obtained from 18, which found the same diameter of inhibition halo. The effective inhibition of the growth of S. aureus provided by this oil can be related to thymol content, which has antimicrobial properties. It is a phenol present in different essential oils with antibacterial activity thanks to its ability to improve the permeability of the membrane of the bacteria47.These results perfectly fit with those reported byT. daenensis may be related to the thymol, as it has good antifungal activity against a wide range of plant pathogenic fungi and food contaminants37. The mechanism of action of thymol has not been fully elucidated, but it is believed that it can damage the cell wall of the fungi or cause their cell wall to decay48.All the other essential oils were not able to inhibit the growth of this fungal strain. Again, the antifungal activity of oil from 18 in their study, used the essential oil from the same plant  and reported an effective inhibition of the growth of these two fungi while49, using the same oil, detected a good inhibition of C. albicans growth. Unfortunately, any essential oil obtained from the other species was capable of inhibiting the growth of these two fungal microorganisms.According to these results,B. subtilis and P. vulgaris was inhibited as well, even if in a lesser extent (14 ± 1 mm) than rifampin (19 ± 0 and 8 ± 0 mm) and gentamicin (30 ± 0 and 24 ± 0 mm) while18 found a remarkable effect of this oil against B. subtilis (43 ± 0 mm).To the best of our knowledge any activity against these two bacterial strains have been detected in previous studies. Further, the growth of T. daenensis against E. coli was confirmed by other researcher, even if the results were quite different and the diameter of inhibition halo varied from 7 to 44 mm50.The efficacy of oil from 23 found a good activity against N. asterotricha, probably due to the presence of oleic acid, stearic acid, and linoleic acid, that have inhibitory activities against S. aureus and other microorganisms53. According to this, it has been previously reported that P. aeruginosa is highly sensitive to essential oils54.Differently,Candida albicans is one of the most common pathogenic fungi capable of causing human infectious, which are often difficult to be threated because of the abuse of antibiotic occurred in the last decades55. The oil from T. daenensis represents a natural, promising alternative for the treatment of these infections and thymol seems to be the main responsible of its efficacy since its ability to penetrate the cell membrane and contribute to the clotting of cell contents56. The MFC of this oil against C. albicans (20 µg/mL) was also low and confirmed its effectiveness as antifungal agent49.P. aeruginosa (MIC 63 μg/mL), according to the results obtained by other authors against H. calycinus, H. sessilifolius. Sh. dysenteriae, K. pneumonia, and S. paratyphi-A, even if rifampin was significantly more effective (MIC 16 μg/mL)58.The strongest activity was found against the Gram-negative 32. These results confirmed the influence of plant habitat and conditions on the composition and activity of the essential oils. The MBCs/MFCs of the essential oil from S. inflata were always higher than MICs, indicating that the ability to inhibit the growth of bacteria was higher than that of killing them.In previous studies any antimicrobial effect was detected using the same oil obtained from plants collected from Isfahan Province, IranT. daenensis, N. sessilifolia, H. incanus, and S. inflata growing in the Daran region of Isfahan (Iran), were obtained and their composition and antimicrobial activity were evaluated. The main common components were thymol, oleic acid, (−)-caryophyllene oxide, α-pinene, 1,8-cineole, palmitic acid, ( +)spathulenol, germacrene D, bicyclogermacrene, phytol, camphor, and borneol, 1,8-cineole and oleic acid, while others were randomly present as a function of the used plants. Essential oil from T. daenensis was the most active as it was able to inhibit the growth of different microorganisms, especially S. aureus and A. brasiliensis. Based on the MICs, the essential oils had low MICs and MBCs/MFCs and good effect on Sh. dysenteriae, P. aeruginosa, E. coli, K. pneumonia and C. albicans, then they can be used as natural and valid agents in agriculture, food, pharmaceutical and cosmetic industries for the treatment of microbial and fungal infections or contaminations.In this study, for the first time, the essential oils obtained from


PIK3CD mutation-related immunodeficiency disease. However, the initial dosages of sirolimus remain undecided. The present study aims to explore initial dosages in pediatric patients with PIK3CD mutation-related immunodeficiency disease. Pediatric patients with this disease were analyzed using the population pharmacokinetic (PPK) model and the Monte Carlo simulation. Body weight and concomitant use of posaconazole were included in the final PPK model, where, under the same weight, clearances of sirolimus were 1 : 0.238 between children without and children with posaconazole. Without posaconazole, the initial dosages of sirolimus were 0.07, 0.06, 0.05, and 0.04 mg/kg/day for body weights of 10–14, 14–25, 25–50, and 50–60 kg, respectively. With posaconazole, the initial dosages of sirolimus were 0.02 mg/kg/day for body weights of 10–60 kg. This is the first attempt to build a sirolimus PPK model for recommending initial dosages in children with PIK3CD mutation-related immunodeficiency disease, thereby providing a reference for individualized clinical drug administration.Sirolimus is used to treat pediatric patients with  PIK3CD mutation-related immunodeficiency stems from autosomal dominant inheritance, leads to hyperactivity of the PI3K/Akt/mammalian target of the rapamycin (mTOR) signaling pathway, induces cell differentiation and proliferation, and results in liver, spleen, lymph node enlargement and other manifestations indicating the occurrence of disease  is often used to detect sirolimus concentration in order to adjust the dosage to reach the required range of treatment window. The next sirolimus dosage is often optimized in light of the drug concentration level to achieve the purpose of synergism and toxicity reduction. However, this dosage adjustment needs to be based on a measurement of existing sirolimus concentration, and so this method is not useful for determining initial dosages as no sirolimus levels are available for reference. Population pharmacokinetic (PPK), combined with a Monte Carlo simulation, provide feasible technical support for solving initial dosage recommendations in clinical practice. In recent years, more and more related research has been reported, which further verifies the reliability and practicability of this method . Hence, PIK3CD mutation-related immunodeficiency disease at the Children’s Hospital of Fudan University  between September 2017 and January 2022 were retrospectively collected. Inclusion criteria were as follows: 1) treated with sirolimus, and 2) carrying out TDM for sirolimus. The trough level of sirolimus concentration was tested using the Emit 2000 Sirolimus Assay . The study was approved by the Research Ethics Committee of the Children’s Hospital of Fudan University . As the study was retrospective, it was approved by the ethics committee of our hospital without the need for written informed consent.Pediatric patients with A first-order conditional estimation method with interaction (FOCE-I method) by NONMEM  was used to build up the PPK model, in which the pharmacokinetic parameters included apparent oral clearance (CL/F), volume of distribution (V/F), and absorption rate constant .i represents the individual parameter value; TV(A) represents the typical individual parameter value; and ηi represents symmetrical distribution, which is a random term with zero mean and variance omega2 (ω2).i represents the observed concentration; Ci represents the individual predicted concentration; and ε1, ε2 represents symmetrical distribution, which is a random term with zero mean and variance sigma^2 (σ2).Equations 2-4 show the residual unexplained variability (RUV), i represents the ith individual parameter; Ei represents the ith individual weight; Estd represents the standard weight of 70 kg; Dstd represents the typical individual parameter, whose weight is Estd; and index represents the allometric coefficient: 0.75 for the CL/F and 1 for the V/F  represents the typical individual parameter value; θ represents the parameter to be estimated; Covi represents the covariate of the ith individual; and Covm represents the population median for the covariate. Potential covariates in the analysis include gender, age, albumin, alanine transaminase, aspartate transaminase, creatinine, urea, total protein, total bile acid, direct bilirubin, total bilirubin, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and co-medications cefdinir, omeprazole, posaconazole, and sulfamethoxazole. Objective function value (OFV) changes are used as covariate inclusion criteria, where the decrease of OFV greater than 3.84 (p < 0.05) is considered sufficient for inclusion in the base model, and the increase of OFV greater than 6.63 (p < 0.01) is considered sufficient for significance in the final model . The model distribution was evaluated by distribution of weighted residuals for model . In addition, the medians and 2.5th-97.5th percentiles of the results from bootstraps  were compared with final model parameters.The model visualization was evaluated using goodness-of-fit plots  PIK3CD mutation-related immunodeficiency disease, based on the final model, and where the target concentration window of sirolimus was 5–15 ng/ml. In the present study, we found weight and concomitant medication of posaconazole affected sirolimus clearance. Therefore, we simulated two different situations based on whether the children were co-administered with posaconazole or not. In each situation, 1,000 virtual pediatric patients with PIK3CD mutation-related immunodeficiency disease were simulated in ten dosages  for six weight groups , respectively. The probabilities of achieving the target concentration window were used as the evaluation criteria, and the probabilities of exceeding the upper limit of the treatment window (15 ng/ml) at 1,000 simulated concentration were used as an evaluation of safety.The Monte Carlo method was used to simulate initial dosages of sirolimus in pediatric patients with PIK3CD mutation related-immunodeficiency disease were included, 16 boys and 8 girls, whose ages were from 2.15–17.92 years and weight from 10–63 kg. The patients having oral sirolimus were receiving it once daily. Demographic data of the pediatric patients with PIK3CD mutation-related immunodeficiency disease and drug combinations are shown in A total of 24 pediatric patients with The RUV model with additive error method was selected. vs. time, and PIK3CD mutation-related immunodeficiency disease without posaconazole, and PIK3CD mutation-related immunodeficiency disease with posaconazole, and PIK3CD mutation-related immunodeficiency disease without posaconazole, the probabilities of exceeding the upper limit of the target concentration were less than 9%, 8%, 6%, and 1%, for the dosages of sirolimus 0.07, 0.06, 0.05, and 0.04 mg/kg/day, respectively. As shown in PIK3CD mutation-related immunodeficiency disease with posaconazole, the concentrations from dosages of 0.02 mg/kg/day sirolimus were all within the treatment window, and there was no value exceeding the upper limit of the target concentration. We simulated two different situations, based on whether the children were co-administered posaconazole or not. PIK3CD mutation-related immunodeficiency disease, which is also known as activated phosphoinositide 3-kinase δ syndrome (APDS)-1, and which is mainly due to an overactivated PI3K/Akt/mTOR signaling pathway, can be treated from the cause using sirolimus, an mTOR inhibitor  isoenzyme CYP3A4, and sirolimus is a substrate of the enzyme (PIK3CD mutation-related immunodeficiency disease has not been reported. The present study is the first exploring this particular group of pediatric patients with PIK3CD mutation-related immunodeficiency disease.In the final PPK model of sirolimus from pediatric patients with e enzyme . Previoue enzyme , and live enzyme . HoweverPIK3CD mutation-related immunodeficiency disease based on the final model, and where the target concentration window of sirolimus was 5–15 ng/ml. Of course, there were differences in the range of sirolimus requirement for different diseases. For example, in kaposiform hemangioendothelioma patients, the target concentration of sirolimus was 10–15 ng/ml (PIK3CD mutation-related immunodeficiency disease, respectively. With posaconazole, the initial dosages of sirolimus were 0.02 mg/kg/day for body weights of 10–60 kg. In addition, the safety of the recommended dosages was also considered in this study. For pediatric patients with PIK3CD mutation-related immunodeficiency disease without posaconazole, the probabilities of exceeding the upper limit of the target concentration were less than 9%, 8%, 6%, and 1%, for the dosages of sirolimus, 0.07, 0.06, 0.05, and 0.04 mg/kg/day respectively. For pediatric patients with PIK3CD mutation-related immunodeficiency disease with posaconazole, the concentrations from dosages of 0.02 mg/kg/day sirolimus were all within the treatment window, and there was no value exceeding the upper limit of the target concentration. Based on these data, we may determine that the current dosing regimens are safe and meet clinical requirements.Further, the Monte Carlo method was used to simulate initial dosages of sirolimus in pediatric patients with 15 ng/ml . In rela15 ng/ml . In chro15 ng/ml . In tube15 ng/ml . In pedi15 ng/ml . We simu2 body surface area >40 kg, and 3 mg/m2 body surface area <40 kg, and a maintenance dose of 2 mg/m2 body surface area >40 kg, and 1 mg/m2 body surface area <40 kg daily, could be effective in treating chronic immune thrombocytopenia in children. Meanwhile, as the limiting condition of narrow therapeutic border and variable bioavailability, sirolimus blood levels were monitored, whose reference range was 5–15 ng/ml (Additionally, in relation to other diseases, 15 ng/ml . AlthougPIK3CD mutation-related immunodeficiency disease, the number of pediatric patients included in the present study is small. In addition, the retrospective study design may influence the target concentrations of sirolimus to some extent. It will therefore be necessary to expand the number of pediatric patients included in future prospective studies to further verify our conclusions.However, this study also had some limitations. Overall, due to the lower morbidity of PIK3CD mutation-related immunodeficiency disease. Without posaconazole, the initial dosages of sirolimus were 0.07, 0.06, 0.05, and 0.04 mg/kg/day for body weights of 10–14, 14–25, 25–50, and 50–60 kg, respectively. With posaconazole, the initial dosages of sirolimus were 0.02 mg/kg/day for body weights of 10–60 kg.This is the first effort to build a sirolimus PPK model and recommend the initial dosages in pediatric patients with


Bacillus cereus is a common environmental foodborne microorganism that is mainly found to harbor toxigenic genes with multiple antibiotic resistances and is linked to threatening the safety of dried milk in concern to powdered infant milk formula. In the current investigation, the mean value of B. cereus in 140 samples of powdered milk was 0.57 × 102 ± 0.182 × 102, 0.15 × 102 ± 0.027 × 102, 0.21 × 102 ± 0.035 × 102, and 0.32 × 102 ± 0.072 × 102 CFU/g in a percentage of 64.0 samples of whole milk powder, 43.3 of skim milk powder, 26.7 of powdered infant milk formula and 36.7 milk–cereal-based infant formula, respectively. The results revealed that B. cereus isolates were found to harbor toxigenic genes in the following percentages: 77.8, 2.0, 72.7, 16.2, and 67.7 for nhe, hbl, cytK, ces, and bceT, respectively. Despite all evaluated B. cereus strains were originated from dairy powders, they showed a significant difference (P < 0.05) in their harbored toxigenic cytK gene between whole and skim milk powders with powdered infant formula and milk–cereal-based infant formula, as well as between powdered infant formula and milk–cereal-based infant formula. All isolated B. cereus strains were resistant to cefoxitin, colistin sulfate, neomycin, trimethoprim–sulfamethoxazole, oxacillin, and penicillin. Based on the antimicrobial resistance of B. cereus strains to cephalothin, chloramphenicol, nalidixic acid, and tetracycline, there was a significant difference (P < 0.05) between powdered infant milk formula and whole milk powder strains. This survey is one of few studies proceeded in Egypt to determine the prevalence of toxigenic B. cereus strains in milk–cereal-based infant formula and powdered infant formula as well as skim milk powder.The online version contains supplementary material available at 10.1007/s00203-022-02945-3. Dairy powders are a prevalent product due to their extended usable time with multilateral kind. Infants are massive consumers of powdered milk-based products, as they may potentially in any way be with a weak immune system, especially newborns that did not receive breastfeeding; the passive immunity was not being transferred to them. Baby foods are well known to be the significant nutrition source of choice for kids, especially in the first part of life when they cannot digest other complex food. Their high values for proteins, minerals, fats, and vitamins are undeniable. Infants and babies have the weakest immune system, so the safety and hygienic quality of these baby foods are highly significant in avoiding and controlling their microbial contamination  or known as B. cereus group with other genetically linked species as B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. cytotoxicus, B. toyonensis, and B. weihenstephanensis  and non-hemolytic enterotoxin (nhe) genes. Other enterotoxins consist of a single protein encoded for bceT (B. cereus enterotoxin), cytK, as well as one emetic toxin (ces) , and 30 dried milk–cereal-based infant formula (complementary food from 6 month age) were collected from various shops, supermarkets, and pharmacies in Cairo and Giza Governorate, Egypt. These products were imported and repacked in Egyptian factories.®400) until complete homogenization. Then, 1 ml of first dilution was distributed and spread on four plates of Mannitol Egg Yolk-Polymyxin agar (MYP)  as follows: 0.3 ml, 0.3 ml, 0.3 ml, and 0.1 ml. All plates were incubated at 32 °C for 24 h. Five presumptive positive colonies had been selected for confirmation. For all isolates, different biochemical tests have been applied as Gram stain, catalase, nitrate reduction, Voges–Proskauer, anaerobic fermentation of glucose, tyrosine decomposition, and lysozyme resistance to confirm which of them belonged to the B. cereus group and other tests to differentiate species of B. cereus s.l., besides evaluation of virulence factors of B. cereus strains as follows: motility test, rhizoid growth, hemolytic activity, growth at 6 ℃ and 50 ℃, crystal protein staining, and starch hydrolysis test. B. cereus ATCC 14,579 and B. cereus ATCC 11,778 were used as reference strains for the biochemical and molecular tests. After biochemical identification, the count of B. cereus was calculated.Twenty-five grams of samples were diluted with 225 ml of 0.1% peptone water  to prepare serial dilutions and mixed in a stomacher . The supernatant contains DNA stored at − 20 °C.Genomic DNA was extracted from the culture positively identified as gyrB gene by primer pair BC1/BC2r and identified as B. cereus using positive control   using multiplex PCR according to protocol mentioned by Ehling-Schluz et al.  between whole milk powder and skim milk powder samples, as well as samples of whole milk powder and powdered infant milk formula , and biofilms formed with spores germinate and attach to plant equipment (such as stainless steel) with even resistance to sanitation. Using raw materials of low spore count and improving routine examination to understand the master step during processing at which contamination with spores happened are confirmed to be effective as control and preventive approaches , 20 for skim milk powder (80.0% B. cereus and 20.0% B. thuringiensis), 16 for powdered infant milk formula  and 30 for milk–cereal-based infant formula . Furthermore, Hwang and Park B. cereus and 58.2% B. thuringiensis from 99 powdered infant formula samples.As presented in Fig. B. thuringiensis is a common pathogen in milk; it has been stated to produce enterotoxins in food and exhibit cytotoxicity  which also have been associated with multiple outbreaks , nearly like data recorded by Sadek et al.  by 16 isolates. The cytotoxic gene has hemolysis and necrosis activities on cells and is responsible for causing fatal poisoning outbreaks  in the following manner: 22 (40.7%), 10 (62.5%), 3 (33.3%), and 6 (30.0%) isolates from whole milk powder, skim milk powder, powdered infant milk formula, and milk–cereal-based infant formula, respectively, while uniquely 3 (5.6%) from whole milk powder isolates harboring 4 genes  and 2 (10.0%) from dried milk–cereal-based infant formula were expressed . Based on the prevalence of nhe and cytK genes, there was a significant difference (P < 0.05) between B. cereus strains isolated from skim milk powder and milk–cereal-based infant formula. In addition, there was a significance difference between B. cereus strains of whole and skim milk powder for ces and bceT genes, as well as between whole milk powder and milk–cereal-based infant formula for ces and cytK gene.As displayed in Figs. S2 and S3, cytK and bceT genes, there was a significant difference between B. cereus strains isolated from powdered infant milk formula and milk–cereal-based infant formula. Besides a significant difference between B. cereus strains on harboring cytK gene between whole milk powders and powdered infant milk formula, no significant difference was found (P > 0.05) in carrying hbl gene for all isolated strains as shown in Table B. cereus strains were found to harbor more than toxigenic genes such as nhe, bceT, and/or cytK, so they may have the possibility to result in emetic and diarrheal food poisoning concurrently. Our findings were almost like the research reported by Rahimi et al. . While Sadek et al. (B. cereus strains from milk-based baby formula to carry enterotoxigenic genes in the following proportions: 95.5% (43) for cytK gene, 71.1% (32) for nhe, and 11.1% (5) for hbl genes.However, Di pinto et al.  reportedk et al.  had revehbl, nhe, and cytK toxigenic genes have caused food poisoning in individuals, as hbl and nhe are responsible for hemolytic and cytotoxic properties, while cytK has been recorded to cause diarrhea with blood .In Fig. nhe, cytK, ces, and bceT genes, in percent of 1.2, 1.4, 6.2, and 1.5, respectively. Although some strains were not hydrolyzed starch and exhibited weak hemolysis on blood agar, they could express enterotoxigenic genes in the low percent, 6.6% nhe, 5.5% cytK, and 1.5% bceT gene, while Organji et al.  between expressing tic Fig. . Some reB. cereus strains, these belonged to skim milk powder that had shown the most similarity in their pattern and followed by whole milk powder strains. All skim and whole milk powder strains were inhibited by chloramphenicol, gentamycin, nalidixic acid, tetracycline, tobramycin, streptomycin, and vancomycin, and it resisted the cefoxitin, cephalothin, colistin sulfate, neomycin, trimethoprim–sulfamethoxazole, oxacillin, and penicillin  strains from milk–cereal-based infant formula were resistant to erythromycin and nine 45.0%) to nalidixic acid  with whole milk powder strains. While based on resistance to chloramphenicol and tetracycline, strains from powdered infant milk formula were significantly different (P < 0.05) from whole milk powder, skim milk powder, and milk–cereal-based infant formula strains. As well as based on the results of B. cereus strains’ resistance to erythromycin, there was a significant difference between whole and skim milk powder strains. In addition, there was a significant difference between B. cereus strains isolated from whole milk powder and milk–cereal-based infant formula referred to their resistance to nalidixic acid and tetracycline antibiotics, as well as between skim milk powder and milk–cereal-based infant formula.As presented in Table B. cereus strains based on antibiotics resistance, strains obtained from powdered infant milk formula and whole milk powder exhibited a higher prevalence statistically significant difference (P < 0.05) than isolated strains from skim milk powder and milk–cereal-based infant formula. There was no significant difference (P > 0.05) in antimicrobial resistance of all obtained B. cereus strains toward cefoxitin, colistin sulfate, gentamycin, neomycin, tobramycin, trimethoprim–sulfamethoxazole, oxacillin, penicillin, streptomycin, and vancomycin.With a comparison of B. cereus isolated from dairy food with the more restricted policy in the utilization of antimicrobials Below is the link to the electronic supplementary material.


Milk and dairy products are sources of exposure to estrogenic endocrine disrupting compounds (e-EDCs). Estrogenic disruptors can accumulate in organisms through the food chain and may negatively affect ecosystems and organisms even at low concentrations. Therefore, the analysis of e-EDCs in dairy products is of practical significance. Continuous efforts have been made to establish effective methods to detect e-EDCs, using convenient sample pretreatments and simple steps. This review aims to summarize the recently reported pretreatment methods for estrogenic disruptors, such as solid-phase extraction (SPE) and liquid phase microextraction (LPME), determination methods including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Raman spectroscopy, and biosensors, to provide a reliable theoretical basis and operational method for e-EDC analysis in the future. According to the U.S. Environmental Protection Agency (EPA), endocrine-disrupting compounds (EDCs) are exogenous agents that can interfere with the synthesis, secretion, transport, metabolism, binding, or elimination of natural blood-derived hormones, which are responsible for homeostasis, reproduction, and development processes . EstrogeDifferent e-EDCs have been identified in milk and dairy products, including endogenous natural estrogens, synthetic estrogens, phytoestrogens, fungal estrogens, and other e-EDCs .−1/day [−1/day [−1 [Endogenous natural estrogens in animals, also known as natural steroid estrogens, include estrone (E1), estradiol (17β-E2 and 17α-E2), estriol (E3), and their glucuronidation and sulfate metabolites. In cows, estrogens are mainly synthesized in the ovaries and placenta. Lipophilic estrogens can easily pass through the blood-milk barrier, making their concentrations in milk directly related to blood levels. Therefore, throughout pregnancy, the concentration of estrogens in milk increases with the levels of estrogens secreted by the placenta . Studies−1/day ,15  and other chemicals such as polycyclic aromatic hydrocarbons (PAH), pesticides, polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), some drugs (such as antiepileptic drugs), fungicides, and cotinine are considered to be e-EDCs . The addE-EDCs enter the human body through water and food intake, inhalation, and skin . These cUnmetabolized e-EDCs or estrogen-active metabolites present in feces can be transferred from field feces to groundwater and finally to surface water . In the The residual levels of e-EDCs in the milk matrix were quite low . The mat−6–5 × 10−6 mg·mL−1, respectively [SPE is the separation of isolates based on the partition coefficient between liquid and solid phases . SPE devectively . An autoectively .SPME is a solvent-free technology widely applied in food, forensics, biomedical, and environmental fields . The pri−1, respectively. The recovery was 75.1–97.2% [3O4@MIL-53(Al) frameworks with HPLC-photodiode array detection. Fe3O4@MIL-53(Al) was used as the MSPE adsorbent to extract three e-EDCs from milk. The recovery rate of the method was 88.17–107.58% [MSPE is a dispersive SPE that applies magnetic adsorbents to bind target analytes. First, the adsorbent is easily separated using an external magnetic field. Suitable solvents were then added to elute the analytes, followed by magnetic separation, and the liquid phase was collected for further analysis . This me.1–97.2% . Yang et.1–97.2% . A novel–107.58% . The mag–107.58% .Currently, LPME is the most widely used sample extraction method. In a simple step, LPME can be used to extract, concentrate, and inject samples. It usually occurs between a few microliters of water-insoluble solvent and the aqueous phase containing analytes . The liqDLLME has become an environmentally benign technique for sample preparation because of its simple operation, high speed, and low consumption of solvents and reagents . DLLME iHF-LPME devices are porous hollow fiber membranes in impregnated with organic solvents, which are based on the use of a supported liquid membrane (SLM) containing the extraction phase and separating it into the sample phase, resulting in a high specific surface area extraction and high enrichment factors . The mos4 and NaCl) for distribution purification. The addition of an appropriate adsorbent can also be purified using SPE, which has the advantages of low cost, simple operation, sensitive detection, and short time consumption [QuEChERS is a simple and direct extraction technology that includes initial segmentation, followed by extraction and cleaning by dispersed SPE . The metsumption . Howeversumption . Xiong esumption . In anotsumption . An imprsumption .Milk contains various proteins, fats, minerals, and carbohydrates, which may reduce detection sensitivity and even lead to detection failure . E-EDCs −1, respectively [−1. Its application in real raw milk samples statistically confirmed the difference in milk between pregnant and nonpregnant cows [−1 in milk using LC-MS/MS. This technique used a C18 column . Mobile phase A and mobile phase B of LC were water and methanol containing 0.1% formic acid, respectively, and the column oven temperature was 40 °C. MS used the positive ESI mode. The levels of 17α-E2, 17β-E2, and E1 were 31, 6, and 159 ng·kg−1, respectively [v/v) and 5 mM ammonium hydroxide/methanol/acetonitrile . Ionization was achieved through negative ESI mode. The LODs of free e-EDCs in milk were 6, 10, 10, and 37 ng·L−1, respectively. Several additional conjugated estrogenic metabolites were found at 2 ng·L−1 for estrone-3-sulfate, 7 ng·L−1 for estrone-3-glucuronide, 6 ng·L−1 for 17β-estradiol-3,17β-sulfate disodium salt, and 7 ng·L−1 for 17α-ethinylestradiol-3-glucuronide. A peak with a nominal mass of molecular ions similar to sulfated E2  was also found during samples analysis. This study used QTOF-MS and LC connected to a LCQ Advantage ion trap (LC–IT-MS) were used to demonstrate that the unknown peak was not an E2-3S isomer [4OH and MeOH:MeCN , respectively, and the column oven temperature was 40 °C. MRM was performed in negative ESI mode. The LOQs were 0.02–0.60 µg·L−1 and 0.02–0.90 µg·kg−1, respectively [−1 [v/v) ammonia) were used for LC using an UHPLC BEH C18 column . In addition, column and sample temperatures were 40 °C and 25 °C, respectively. Analysis was performed in negative ESI mode with higher sensitivity and stability. The LOQ of the method was 0.003–0.015 µg·kg−1 [LC-MS is a combination of two selective techniques that allows the isolation and measurement of analytes of interest in highly complex mixtures, such as proteins or peptides, and simultaneously detects specific compounds according to their elemental and structural characteristics. LC distinguishes the physicochemical properties of compounds, whereas MS distinguishes the masses of compounds  ,137. BPAectively . To evalectively . A similant cows . Anotherectively . The comS isomer . Socas-ReOH:MeCN :50, v/v, µg·kg−1 .−1 [−1 [−1, respectively [−1, and the LOQs were 0.16–6.11 and 0.11–4.86 µg·L−1, respectively. The LOD and LOQ of natural yogurt were 0.07–3.73 and 0.23–9.81 µg·L−1, respectively [GC-MS is widely used, because it has good selectivity, high separation degree, high sensitivity and repeatability, and relatively stable in the process of use. The two ionization forms of GC-MS are electron ionization (EI) and chemical ionization (CI). In addition, GC-MS is easy to use and provides insight into the identification of compounds . Scienti−1 . A simil−1 [−1 . The majectively . An enviectively .At present, biosensors are widely used in the fields of biomedical diagnosis, immediate monitoring of therapeutic and disease progress, food control, drug discovery, forensics, and biomedical research, and are also regarded as analytical tools . Biosens−1, respectively. This method was validated using milk powder, and the results displayed high accuracy [−1, and the LOD is 0.2 pg·mL−1, which show good adaptability in the milk sample [3O4 nanoparticles. Under the optimal conditions, the linear range of the sensor pair E2 was 10−11–10−6 M, and the LOD was 3.48 × 10−12 M. The sensor showed good selectivity and repeatability for the analysis of E2 in milk [−14–1.0 × 10−9 mol·L−1, and the LOD was 7.2 × 10−15 mol·L−1 [−1 with a LOD of 0.2 nmol·L−1. The results from spiked milk showed good recovery and repeatability [Pan et al. used an indirect competitive immunoassay to prepare an electrochemical immunosensor that can simultaneously detect four phenolic estrogens: HEX, DES, DS, and BPA. Differential pulse voltammetry was applied and the amperometric response sequence was EDS > DS > BPA > HEX, and the LODs were 0.25, 0.15, 0.20, and 0.25 ng·mLaccuracy . A simplaccuracy . A fluork sample . A novel in milk . Au nano mol·L−1 . An unla mol·L−1 . Karthiktability .−1, which is lower than the level of progesterone in commercial milk and randomly purchased raw milk [−1. These results are consistent with those of ELISA [−1 [−1, respectively [−1, and their LODs were 7.8 and 92 pg·mL−1, respectively. The capture time was reduced to 10 min [−1, and it is successfully applied to detect E2 in milk samples [−1 [2+ dependent DNAzyme was developed. The sensor employed an anti-BPA aptamer as the recognition element for BPA binding. The double-labeled substrate DNA was cleaved into two segments by synergistic DNA hybridization to form an Mg2+-dependent catalytic DNAzyme. The separation of the fluorophore and quencher resulted in a high fluorescence response to BPA determination, and the LOD was 50 fM in milk samples [A biosensor based on total internal reflection fluorescence was developed to detect progesterone in milk samples. The LOD of progesterone in UHT milk, fresh milk, and raw milk by the method was 45.5–56.1 pg·mLraw milk . Daems eof ELISA . A novelLISA [−1 . A novelectively . Ren et o 10 min . An indi samples . An immuples [−1 . A BPA f samples .−15 to 1 × 10−9 M under optimal conditions, and the LOD was 3.3 × 10−16 M [−1, and the BPA in liquid milk samples has been successfully detected [Photoelectrochemical sensing technology has attracted increasing attention in the field of biological and environmental molecular detection owing to its high sensitivity, low cost, and ease of miniaturization. Quantitative detection is typically based on photoelectron transfer at the electrode/electrolyte interface and photocurrent changes due to the interaction between the target and the photoactive matrix or probe . A highl 10−16 M . Qiao etdetected .−1, with sufficient sensitivity for the detection of 17β-E2 in milk [−1. In quantitative analysis, the proposed method showed a good dynamic linear response at logarithmic concentrations [−1, and the levels of ochratoxin A and ZEN were lower than the LOD (<5 ng·L−1) [−1. The mean concentration was 3.41 ± 0.13 (mean ± SD) ng·mL−1 [ELISA is a quantitative assay that displays antigen-antibody responses by color changes of enzyme-linked conjugants and enzyme substrates and is used to determine the presence and concentration of target molecules . Bai et  in milk . A plasmtrations . Samiee  ng·L−1) . Kuruto- ng·mL−1 .2+ as polymerization agents was established to detect BPA residues in milk with high sensitivity. Under optimal conditions, the LOD of BPA was 4.3 × 10−9 moL·L−1 (0.98 × 10−3 mg·kg−1), lower than the European Standard (0.6 mg·kg−1) [−4 M. Each Raman spectroscopy fingerprint of azo dye is a specific hormone. This technique can be used not only for the detection of phenolic hormones in coupling reactions, but also in the study of other phenolic molecules [−1 [Raman spectroscopy is rarely used in the study of estrogens, mainly because of its inherently weak signals and fluorescence interference caused by fluorescent pollutants in most samples. Special Raman techniques such as SERS are typically required . SERS ismg·kg−1) . An easyolecules . A simplules [−1 .−1 by improving the pre-processing steps or innovative detection methods. The current pre-processing and detection technologies that are applied in milk and dairy products are either expensive or complicated to operate. HPLC and GC not only need professional operators, but also cannot be used for on-site testing. Electrochemical sensors and SERS require too much time during sample preparation and detection and are not widely used in commercial detection. SPE in pretreatment methods and ELISA, LC-MS/MS, and GC-MS in detection methods are widely used. In addition, real-time monitoring should be performed on how e-EDCs enter raw milk, and the detection instruments and conditions can be optimized to make them portable and green and improve the detection efficiency. Based on the above problems, the development of portable, automatic, and low-cost pretreatment and detection technology should be strengthened in the future.At present, milk and dairy products are common foods in people’s daily lives; however, they are also a major source of estrogen disruptors. E-EDCs have become a global health problem, posing a threat to human health. Trace e-EDCs enter the human body and cause potential harm through accumulation, including damage to the endocrine system. Therefore, fast, sensitive, and efficient methods are needed to detect multiple low-dose and ultra-low-dose e-EDCs. In this review, the principles of LC-MS/MS, GC-MS, estrogen biosensors, ELISA, Raman spectroscopy, and other detection methods are introduced, and sample pretreatment is summarized. In this regard, many techniques can reach the detection level of ng·L


Paracoccus sp. KKU01. A maximum biomass concentration of 55.5 g/L was attained using the C/N ratio of 10, initial sugar concentration of 100 g/L, and 20% DO set point. Fed-batch cultivation conducted under these optimum conditions, with two feedings of SJ-based medium, gave the final cell concentration of 87.9 g/L, with a PHB content, concentration, and yield of 36.2%, 32.1 g/L, and 0.13 g/g-sugar, respectively. A medium-based economic analysis showed that the economic yield of PHB on nutrients was 0.14. These results reveal the possibility of using SJ for high cell density cultivation of Paracoccus sp. KKU01 for PHB production. However, further optimization of the process is necessary to make it more efficient and cost-effective.High cell density cultivation is a promising approach to reduce capital and operating costs of poly (3-hydroxybutyrate) (PHB) production. To achieve high cell concentration, it is necessary that the cultivation conditions are adjusted and controlled to support the best growth of the PHB producer. In the present study, carbon to nitrogen (C/N) ratio of a sugarcane juice (SJ)-based medium, initial sugar concentration, and dissolved oxygen (DO) set point, were optimized for batch cultivation of  Cane sugar is an important agro-industrial product of many countries, including Thailand. Due to its suitable climate and landscape , Thailan4H6O2 (Poly (3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible bioplastic belonging to the group of polyhydroxyalkanoates (PHAs). It is a natural polyester, with a repeating unit C4H6O2 , that ca4H6O2 . This bi4H6O2 , ranging4H6O2 . Due to Cupriavidus necator is known to be one of the most promising ones, owing to its ability to consume a wide range of substrate, high growth rate, and high PHB production  hydrolysate  ratio of the SJ-based medium, initial total sugar concentration, and dissolved oxygen (DO) set point, were optimized to achieve the best cell growth. The optimum conditions were then used in fed-batch fermentation to produced PHB. Based on the results, a medium-based economic analysis was performed and the feasibility of PHB production by Paracoccus sp. was discussed.To date, hundreds of microorganisms have been reported to be able to produce PHB. Among others, oduction . Howeveroduction . Therefoglycerol , methanoglycerol , and sucglycerol , for PHB necator . It can  necator , and hasrolysate . PHAs prrolysate , pure glrolysate , and def biomass . However biomass . The resca. 600 g/L, and stored at −20°C until use. The concentrated sugarcane juice (SJ) contained sucrose as the major component. Potassium, magnesium, and calcium were the major elements in the concentrated SJ. Protein was also present in the juice, but at a very low level. The compositions of the concentrated SJ are given in Freshly harvested sugarcane stalk was purchased from local farmers in Khon Kaen, Thailand. It was milled to obtain the juice, which was subsequently concentrated by boiling to a total sugar concentration of Paracoccus sp. KKU01 was used as the PHB producer. It was isolated from a soil sample collected at a cassava starch production plant, Kaensiri Starch Co., Ltd., Phra Yuen, Khon Kaen, Thailand. The bacterium was screened and isolated using the method of Paracoccus denitrificans (Accession no. MT491243). Preliminary study showed that Paracoccus sp. KKU01 could utilize glucose and sucrose for growth, and could accumulate PHB under nitrogen-limited conditions (unpublished data). The bacterium was maintained on nutrient agar, supplemented with 20 g/L of sucrose as the carbon source, at 30°C. Inocula were prepared by transferring one loop-full of the bacterium growing on nutrient agar into nutrient broth containing 20 g/L of sucrose, and incubating at 30 °C, 150 rpm, pH 6.9 for 15 h. PHB production medium was prepared following the formula of 2HPO4, 0.5 g of KH2PO4, 1 g of MgSO4, and 2 ml of trace element solution. SJ  and (NH4)2SO4 were used as the carbon and nitrogen sources, respectively. The trace element solution consisted of (per 1 L of 1 N HCl) 10 g of CaCl2·2H2O, 4.98 g of FeSO4·7H2O, 0.23 g of ZnCl2, 0.78 g of CuSO4·5H2O, 0.24 g of Na2MoO4·2H2O, and 0.61 g of MnSO4·H2O. The SJ, MgSO4, and trace element solution were sterilized separately at 121°C for 15 min, and aseptically mixed after being cooled to room temperature.4)2SO4 to adjust the C/N ratios to 4 to 53 (by mass). The C/N ratio of four was selected based on the elemental compositions of Paracoccus sp. (CH1.8O0.48N0.25) reported by Paracoccus denitrificans . Effects of the initial sugar concentration in a range 30–150 g/L was subsequently examined using the optimum C/N ratio. The cultivation conditions were the same as described above. Samples were taken to follow the bacterial growth (in terms of OD600), and the consumption of total sugar during the fermentation. The experiments were conducted in triplicate, and the mean and standard deviation of the mean are reported.Effects of carbon to nitrogen (C/N) ratio and initial sugar concentration were investigated in 500-mL Erlenmeyer flasks, with a working volume of 100 mL, using one-factor-at-a-time approach. In C/N ratio optimization, concentrated SJ was diluted with distilled water to a total sugar concentration of 30 g/L. Then, the diluted SJ was supplemented with minerals according to the mineral medium formula, and added with calculated amounts of , with a working volume of 1 L and an aeration rate of one vvm. The DO set point was varied in a range 10–50% of the oxygen saturation value. DO concentration was maintained at the designated level by automatic control of the agitation rate in the range 300 to 1,500 rpm. The bioreactor containing sterile medium was inoculated with Paracoccus sp. culture to an initial OD600 of 0.2 unit, and the fermentation was carried out at 30 ± 2°C, and pH 6.9 ± 0.2 (controlled using 10 N NaOH and 10 N HCl). Samples were taken regularly for the determination of cell dry mass (CDM) and total sugar concentrations.Using the optimum C/N ratio and initial total sugar concentration, effects of dissolved oxygen (DO) level on growth of 4)2SO4 to attain a C/N ratio of 10) was fed into the bioreactor to increase the sugar concentration to around 100 g/L. The feeding of the medium was performed twice. The final volume of the medium was approximately 1 L. Samples were taken at 6-h intervals for the determination of CDM, total sugar, ammonia-nitrogen (NH3-N) and PHB concentrations. This experiment was conducted in duplicate.Fed-batch fermentation was carried out in the 2-L bioreactor with an initial volume of 0.8 L. The fermentation temperature, pH, and DO set point were controlled at 30 ± 2°C, 6.9 ± 0.2, and 20%, respectively. The experiment was started as a batch fermentation with the optimum initial total sugar concentration (100 g/L), and was allowed to proceed until the sugar concentration decreased to below 30 g/L. Then, calculated amount of fresh medium (SJ that was previously supplemented with (NHEY), defined as monetary unit (USD) of PHB produced per monetary unit (USD) spent in nutrients, was calculated using Y is the concentration of PHB (kg/L), C is the cost of each nutrient used in medium preparation (USD/kg), N is concentration of each nutrient (kg/L), and I is the selling price of PHB (USD/kg)  . It shou(USD/kg)  was usedalfa.com .EY=Y∑. CDM (g/L) was measured gravimetrically after washing the cells twice with distilled water, and drying at 80°C until a constant weight was reached. Residual cell dry mass (RCDM) concentration was calculated as the difference between CDM and PHB concentrations. Total sugar concentration was determined using the phenol-sulfuric method , Rm is the maximum rate of CDM, RCDM, and PHB production (g/(L·h)), e is the Euler’s number (2.7183), λ is the lag time for the production of CDM, RCDM, and PHB (h), and t is the fermentation time (h). The Solver function of Microsoft Excel 2019 was used for curve fitting and estimation of the kinetic parameter values.Cell growth in terms of ODc method , using se method , using ae method . The PHBe method , where Yµ) during the first 9 h revealed that the bacterium grew equally well at all the C/N ratios tested; the value of µ varied in a narrow range of 0.424–0.484 1/h (ca. 1.0 g/L of (NH4)2SO4 was used in the medium . It could, therefore, be possible that most of the nitrogen was consumed during the exponential phase, resulting in insufficient nitrogen for the later stage of growth. As for the growths at other C/N ratios, both the µ and maximum OD600 values were very similar. It was initially presumed that C/N ratio of four would be suitable for cell growth since the C/N ratio of Paracoccus sp. has been reported to be around 4 2SO4) was used compared with C/N ratio of 4. This was consistent with a report of Paracoccus sp. LL1.Determination of total sugar concentration revealed that sugar was rapidly consumed when the cells were grown at C/N ratios of 4 and 10, and this decreased with increasing C/N ratio up to 53 . It is w and RNA . Therefo and RNA , C/N ratParacoccus sp. KKU01 was grown at initial sugar concentrations of 30–150 g/L. 4)2SO4] concentrations, which can suppress cell growth , e.g., superoxide (O2−) and hydrogen peroxide (H2O2), inside the cells. These ROS can be accumulated to a concentration that overwhelms the antioxidant defenses of the cells, leading to cell inactivation. Some enzymes, particularly dehydratases and metalloenzymes, have also been reported to be inactivated by the presence of superoxide in the system 2SO4) as the feeding solution. Provision of nitrogen source in the feed has been shown to be beneficial for PHB production in several PHB producers, e.g., Pseudomonas extorquens sp. K. .Using the C/N ratio, initial sugar concentration, and DO set point of 10, 100 g/L, and 20%, respectively, ectively . PHB wasectively . Therefoectively . For this sp. K. , RalstonRLB14690 , WautersRLB14690 , CupriavSMAA1020 , Halomon venusta , and Par sp. LL1 . Noted tccus sp. . VigorouParacoccus sp. According to a report of Paracoccus sp. LL1 in a membrane bioreactor. Additionally, secondary metabolites such as polyketides, bacteriocins, and non-ribosomal peptides might also be produced  (4H6O2 (molecular weight 86 g/mol)  , and the6 g/mol) , carbon Cupriavidus necator could be grown to high cell concentrations (148–164 g/L) and produced PHB at high concentrations (109–125 g/L) and productivities [1.5–3.1 g/(L·h)]. Nevertheless, Paracoccus sp. LL1 could also be grown to a high cell density (113 g/L) using a continuous fermentation process with a cell retention system (Paracoccus sp. KKU01 was grown to a high cell density of 87.9 g/L and produced 32.1 g/L of PHB. These figures, although not the highest, were in the upper range among the results compared, suggesting that Paracoccus sp. KKU01 is a potential PHB producer from SJ. Better still, the bacterium may be used with other low-cost feedstocks containing sucrose, e.g., cane molasses (EY calculation revealed that the process used in the present study gave a low EY of 0.14, indicating that the revenue of PHB production is less than the cost of the medium. It is therefore necessary to further optimize the process to increase PHB production and to reduce the cost of medium. Increasing PHB production may be achieved by precise control of oxygen supply during the process (pha genes involved in PHB synthesis (C. necator DSM 545 (Burkholderia sacchari LMG19450 on xylose (Halomonas boliviensis (EY calculation showed that the cost of K2HPO4 accounted for over 80% of the total medium cost, medium optimization is worth the investigation. SJ already contains appreciable concentrations of potassium and phosphorus as shown in 2HPO4 in the medium to reduce cost of medium preparation.The key results for PHB production from the present study along with those from recent publications are shown in n system , though molasses  and fruimolasses , which w process , by the ynthesis , or by uynthesis . The use DSM 545 , while tn xylose . Double iviensis . HoweverParacoccus sp. KKU01 to a high cell density for PHB production. The growth conditions, i.e., C/N ratio of SJ-based medium, initial sugar concentration, and DO set point were optimized, yielding the maximum biomass concentration of 55.5 g/L at C/N ratio of 10, initial sugar concentration of 100 g/L, and 20% DO set point. Fed-batch cultivation carried out using the optimum conditions gave a final biomass concentration of 87.9 g/L, with PHB content, concentration, and yield of 36.2%, 32.1 g/L, and 0.13 g/g, respectively. Overall, the present study demonstrates the feasibility of using SJ for high cell density cultivation of Paracoccus sp. KKU01 for PHB production. However, the medium-based economic analysis indicates the needs for further optimization of the process, so as to make it more efficient and cost-effective.SJ was used as the sole carbon source to grow


Malaria is a complex issue due to the availability of few therapies and chemical families against Plasmodium and mosquitoes. There is increasing resistance to various drugs and insecticides in Plasmodium and in the vector. Additionally, human behaviors are responsible for promoting resistance as well as increasing the risk of exposure to infections. Chalcones and their derivatives have been widely explored for their antimalarial effects. In this context, new derivatives of chalcones have been evaluated for their antimalarial efficacy.P. berghei NK-65. The efficacy of the three most potent chalcone derivations  identified after an in vitro compound screening test was tested. The selected doses of 10 mg/kg, 20 mg/kg, and 10 mg/kg were studied by evaluating parasitemia, changes in temperature, body weights, organ weights, histopathological features, nitric oxide, cytokines, and ICAM-1 expression. Also, localization of parasites inside the two vital tissues involved during malaria infections was done through a transmission electron microscope.BALB/c mice were infected with p < 0.001) reductions in parasitemia levels on the fifth and eighth days of post-infection compared to the infected control. These derivatives were found to modulate the immune response in a P. berghei infected malaria mouse model with a significant reduction in IL-12 levels.All three chalcone derivative treated groups showed significant (P. berghei.The present study indicates the potential inhibitory and immunomodulatory actions of chalcones against the rodent malarial parasite The online version contains supplementary material available at 10.1186/s12906-022-03777-w. Plasmodium falciparum is the most common Plasmodium species that causes deadly malaria -2-propen-1-one, identified as the most active by in vitro tests, and was tested in mice infected with P. berghei (ANKA), a chloroquine-susceptible strain of murine malaria. This compound was able to decrease the parasitemia and delay the progression of malaria but did not eradicate the infection  from ABCAM, USA and the BD CBA Mouse Soluble Protein Flex Set System for IL-1, IL-6, TNF-alpha, IFN-γ, IL-10 and IL-12p70 were purchased from BD Biosciences, USA. All other chemicals and reagents used in this study were of analytical grade.Plasmodium berghei NK-65 was procured from Punjab University, Chandigarh and was maintained in Swiss albino mice by serial passage by intraperitoneal injections of 0.2 mL of infected blood suspension containing 1 × 106 parasitized red blood cells (pRBC) every 10 days. Furthermore, this strain was used in model validation.7P. berghei NK-65 (chloroquine sensitive strain) infected erythrocytes. Each mouse was checked for any parasitemia, physical signs, and mortality. The parasitemia in all the infected mice was evaluated using a thin smear of peripheral blood taken from the tail followed by Giemsa staining  and visualised under a light microscope with 1000X magnification [Inbred BALB/c, 6–8-week-old mice were inoculated by intraperitoneal injection of 0.2 mL of infected blood suspension containing 1 X 10fication . The finDoses of each chalcone derivative 1 (10 mg/kg), 2 (20 mg/kg), and 3 (10 mg/kg) were obtained after extrapolation of in vitro data published in our previous study , and theA total of thirty-six BALB/c mice were recruited for the main experiment. These mice were randomly divided into six groups Groups 1–6). Each group consists of six mice : Injected intraperitoneally with blank media (PBS).7P. berghei NK-65 infected erythrocytes.Group 2: (Infected): Inoculated by intraperitoneal injection with 1 × 10Group 3: Infected mice treated with CQ.Group 4: Infected mice treated with Chalcone derivative 1.Group 5: Infected mice treated with Chalcone derivative 2 and,Group 6: Infected mice treated with Chalcone derivative 3.rd day of post-infection, which was continued till the 7th day following standard Rane’s test procedure [For the intraperitoneal/oral administration, a suspension formulation of screened chalcones was prepared by triturating the weighed quantity in a dry mortar with 0.5% carboxymethyl cellulose (CMC) and was prepared by drop-wise addition of water and proper grinding, and chloroquine (CQ) was dissolved in PBS (pH 7.2). For all drug/derivatives treated groups, the first administration of the drug/derivatives was started on the 3rocedure . Thin blrd, 5th, and 8th days after inoculation. The percentages of parasitemia were calculated by counting the number of parasite-infected erythrocytes per 2000 erythrocytes according to the equations below [Parasitemia was monitored by light microscope examination under (1000X) magnification using Giemsa-stained blood smears on the 3ns below .rd, 5th, 7th, and 8th days of post-infection [The rectal temperature and body weight of each mouse in all the groups were measured before infection (day 0) and on the 3nfection .The wet weight of two organs, i.e., the liver and spleen of each mouse in all the groups, was measured after sacrifices post-treatment.At the end of the study, the blood samples from each mouse were withdrawn by tail vein. Serum was separated and the nitric oxide levels were measured in serum samples of mice. Nitrite is estimated using the Greiss reagent and serves as an indicator of nitric oxide production. Briefly, 100 µL of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% napthylamine diamine dihydrochloric acid in water) was added to 100 µL of serum, and the absorbance at 546 nm was measured after 15 min . The nitWhole blood was collected in sterile tubes at the post-treatment stage and allowed to coagulate for 2–3 h at 40◦C and then subjected to centrifugation. The sera thus obtained were stored at -80ºC until cytokine measurement was performed by using the BD CBA Mouse Soluble Protein Flex Set System for IL-1, IL-6, TNF-, IFN-γ, IL-10, and IL-12p70 following the manufacturer’s recommendations.in toto for 48 h in a neutral buffered 10% formalin solution. The organs were cut into serial cross sections and fixed in formalin for another 12 h. Finally, tissue sections were fixed in paraffin and cut into 5 mm thick sections, which were stained with haematoxylin and eosin and looked at for inflammation and parasites [Liver and spleen from all experimental groups were removed aseptically after being fixed arasites .2O for 5 min, and slides were dried and mounted with DPX.Tissue sections of the liver and spleen were immunostained as described previously . The Avi3 were fixed in 3% glutaraldehyde-0.1 M sodium phosphate buffer (pH 7.2), for 2 h at 4 °C. All samples were rinsed three times and kept overnight at 4 °C in a 0.2 M sucrose-0.1 M phosphate buffer (pH 7.2). The samples were then post-fixed in 2% OsO4 in 0.1 M phosphate buffer, washed twice in 0.1 M phosphate buffer and twice in distilled water, dehydrated in a graded alcohol series and then in propylene oxide, and embedded in Epon. Using a Reichert-OmU-2 ultramicrotome and a glass or diamond knife, thin slices were cut and deposited on Formvar carbon-coated grids. The sections were poststained with 6% aqueous uranyl magnesium acetate and Reynold’s lead citrate. The section was then examined with a Phillips 200 or 400 transmission electron microscope at 60 or 80 kV, according to the thickness of the section and the required magnification.Ultrastructure changes after the treatment can be visualised by electron microscopy as described previously . The spep < 0.05 was taken as the level of significance. For evaluating the ICAM-1 expression, the Chi-square, contingency co-efficient, and correlation tests were applied.Data is shown as mean ± SEM and analysed using the one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparison test. The analysis was performed using SPSS Version 16.0 software and P. berghei CQ-sensitive NK-65 parasitized RBCs by intraperitoneal injection on the first day (D0). Seventy-two hours later (D3), the mice were randomly divided into five groups of six mice each, and one more additional group of six mice without infection. Three groups of mice were treated with chalcone derivative 1 , derivative 2 , and derivative 3 , respectively. The negative control group was treated with PBS, while the positive control group was treated with CQ (10 mg/kg), respectively. Each chalcone derivative and the standard drugs were treated once daily for five days, i.e., from day 3 to day 7. Giemsa stained thin smears were prepared from tail blood samples collected on day 3rd, 5th, and on 8th days post-infection to monitor parasitemia level. The body weight and rectal temperature were recorded on the 3rd, 5th, 7th and 8th days of post-infection.Using the method described by Ryley and Peters (1970) , the schp < 0.001) reduction in parasitemia levels on the 5th and 8th days of post-infection, including the CQ-treated group compared to the infected control. The percentage parasitemia of derivative 1, 2, 3, and CQ-treated groups were 10.57 ± 2.12, 13.68 ± 1.98, 16.31 ± 3.88, and 9.04 ± 0.55 on the 8th day post-infection as compared to the infected control (39.9 ± 1.8), , CQ, and derivative 2 treated groups significantly (p < 0.05) increased on the 3rd day of infection, and a significant (p < 0.01) increase was also shown in derivative 1 and derivative 2 in temperature on the 7th day post-infection compared to the non-infected control  and (24.8 ± 1.16) till day 8th (24.1 ± 0.87) and (25.36 ± 0.93), whereas a gradual decrease in body weight was observed in infected mice, derivative 1, 2, and 3 treated groups  in the infected control (1.38 ± 0.041) and in the derivatives 1 (1.35 ± 0.03), 2 (1.34 ± 0.12), and 3 (1.33 ± 0.05) treated groups as compared to the non-infected control (0.97 ± 0.02) except for the CQ (1.2 ± 0.3) treated group. Similarly, as compared to the non-infected control group, there was a significant (p < 0.001) increase in the weight of the spleen of the infected control, derivative 1, 2, and 3 treated groups. However, a significant (p < 0.01) reduction in the weight of the spleen was observed in all treated groups as compared to the infected control , IL-10 (p < 0.05), IL-12p70 (p < 0.001), IL-1β (p < 0.01) and IFN-γ (p < 0.01) in infected-control except TNF, which showed non-significant increase as compared to non-infected control. There was a significant (p < 0.001) reduction in IL-6 levels in all treated groups as compared to the infected control. The expression of IL-10 and TNF was found to be statistically non-significant as compared to the infected control in all treated groups. The level of IL-12p70 in the CQ-treated (0.76 ± 0.46 pg/mL), derivative 1 (0.36 ± 0.36 pg/mL), and derivative 3 (0.6 ± 0.39 pg/mL) treated groups were significantly lower (p < 0.001) when compared to the infected control (77.58 ± 15.31 pg/mL) while no expression was observed in the derivative 2 treated group. The level of IL-1β was significantly decreased in derivative 1 (p < 0.01), derivative 2 (p < 0.05), and derivative 3 (p < 0.01) treated, as compared to the infected control. IFN-γ levels, on the other hand, were only significantly lower in CQ-treated groups when compared to the infected groups  analysis revealed significant increase in serum level of cytokines IL-6 . In mild expression, hepatocytes did not show any ICAM-1 expression and it was only partially expressed in the endothelial lining of sinusoids. Marked ICAM-1 expression was observed in all infected control mice with intense positivity on sinusoidal endothelium as well as hepatocytes .Mild ICAM-1 expression in immunostained liver sections was observed in all non-infected control mice Fig. . I B, fotes Fig. . II B foNumerous pRBC were seen adhered to macrophages and endothelial cells via their surface knobs in the liver and spleen in both the CQ treated group and the chalcone derivative treated  under in vivo conditions [P. berghei and P. yoelii, and controls P. berghei infection in rats when administered either orally, intraperitoneally, or subcutaneously once per day for 5 days starting 2 h after infection [The in vivo model was recruited for the present study since it demonstrates considerable prodrug effects and also possible engagement of the immune system in the suppression of infection . P. berg malaria . Further malaria . Rane’s  malaria . In thisnfection . Howevernditions , 46. Simnfection . The prenfection , 47, 48.th day.Body weight loss and body temperature deterioration are the typical attributes of malaria-infected mice . So, ideSplenomegaly and hepatomegaly are among the common phenomena , 53. BotChalcone derivatives have been extensively known for inhibiting NO synthesis, iNOS and cycloxygenase 2 protein expression in lipopolysaccharide stimulated cells. The structure–activity analysis illustrated that chalcones imposed with substituents that can decrease the electronic density in the B ring, such as chlorine atoms or nitro groups, show much better biological activity and selectivity in the inhibition of nitrite production, and mostly position 2 in ring B seems to be more important . Howeverth day post-infection, showing an association with disease severity that supports previous findings. Furthermore, when compared to the infected-control group, chalcones treated groups  and CQ showed significant reductions in IL-6 and IL-1 levels, while other cytokines such as IL-1 and IFN- γ levels were comparatively low in CQ treated and derivative 1 treated groups, indicating protection against disease progression and modulating the immune response in the P. berghei infected malaria mouse model. Also, a significant reduction in IL-12 levels in all treated groups may be associated with a delay in disease progression, which has been found to be increased in the case of uncomplicated malaria [P. falciparum infection [Further, malaria infections are complex syndromes involving a variety of inflammatory responses that can improve cell-to-cell interaction (cytoadherence), cell stimulation with malaria-derived antigens and toxins, and host-derived factors such as cytokines. Moderate levels of cytokines are good for the host as they cause fever . The pre malaria , 59. Elenfection , 61, whinfection . These rnfection .Inflammatory mediators as well as parasite sequestration may be responsible for the disease severity. It has been suggested that the cytokines up-regulate the expression of adhesion molecules such as ICAM-1 that are involved in the binding of the pRBCs to the vascular endothelium . In the Additionally, pathological changes in these infected murine models before and after treatment were observed microscopically in H&E stained slides to depict a clear picture of disease manifestation. In malaria, the main organs infected are the spleen and liver, which mark the pathophysiology of the disease. Here, we have observed an enlarged, oedematous, and brown, grey, or black colored liver, which may be as a result of the deposition of malaria pigment (hemozoin) in the pulp histiocytes and sinusoidal lining cells of the spleen, and mainly in Küpffer’s cells in the liver section, demonstrating the role of macrophages in parasite clearance. There was also phagocytosis of pRBC and RBC by Küpffer’s cells, endothelial cells, and sinusoidal macrophages. Malarial parasites proliferate inside the RBCs of visceral organs, chiefly in the spleen and liver, and this leads to annihilation of RBCs and loss of cells. The parasite's multiplication also results in the synthesis of pigment, which is left over after the parasite digests the cytoplasm of RBCs. As a result, pigment builds up inside these organs, causing them to darken in color.P. falciparum is the only species that is sequestered in deep vascular beds as the intraerythrocytic parasite matures to the trophozoite and schizont stages. By doing so, it escapes the clearance mechanisms of the spleen and is thus able to proceed to schizogony. Sequestration results from the cytoadherence of erythrocytes infected with trophozoites and schizonts of P. falciparum to vascular endothelium; this cytoadherence requires an interaction between specific parasite ligands and endothelial cell receptors [Besides, eceptors . TherefoP. berghei (Fig. P. falciparum. In addition, ICAM-1 expression may play a role in the cytoadhesion of malaria-infected red blood cells or in the recruitment of leukocytes to endothelial activation sites [Chalcones have a vast number of bioactive molecules with a wide range of molecular targets. Even slight structural alterations in chalcones can cause them to target different biological functions. Chalcones are a class of chemicals that could potentially be used to generate low-cost, synthetic antimalarial drugs in the future. The present study clearly indicates the potential inhibitory and immunomodulatory action of chalcones against the malarial parasite hei Fig. , reportion sites . A decreAdditional file 1.


Lactobacillus plantarum R9 and Lactobacillus gasseri B1-27, isolated from the human milk of healthy parturients and the traditional medicine and food homologous ingredient Poria cocos, were separately investigated.To develop functional foods with traditional medicines and homologous food ingredients as well as human milk-derived probiotics, the co-fermentation process of two probiotics, Poria cocos fermentation broth at 2.5% significantly enhanced the total number of L. plantarum R9 (p = 0.001) and L. gasseri B1-27 (p = 0.013) after 20 h of fermentation, and Non-targeted metabolomics assays conducted before and after fermentation of the human milk-derived L. plantarum R9 and L. gasseri B1-27 using the 2.5% Poria cocos fermentation broth revealed 35 and 45 differential metabolites, respectively. A variety of active substances with physiological functions, such as L-proline, L-serine, beta-alanine, taurine, retinol, luteolin, and serotonin, were found to be significantly increased. Mannitol, a natural sweetener with a low glycemic index, was also identified. The most significantly altered metabolic pathways were pyrimidine metabolism, pentose phosphate, yeast meiosis, ABC transporter, insulin signaling, and mineral absorption, suggesting that co-fermentation of human milk-derived probiotics and Poria cocos may affect the metabolism of trace minerals, sugars, organic acids, and amino acids.The Poria cocos to be used in co-fermentation was 2.5% and identified more than 35 differentially expressed metabolites in each probiotic bacteria after co-fermentation. Moreover, several beneficial metabolites were significantly elevated as a result of the co-fermentation process indicating the valuable role of Poria cocos as a functional food.Overall, we determined that the optimal concentration of  Consumers’ increasing attention toward a healthy diet often leads them to consume functional foods, such as probiotic functional milk beverages rich in plant ingredients , which cPoria cocos is the dried sclerotia of Poria cocos (Schw.) Wolf, a fungus belonging to the family of the polyporaceae, is a well-known traditional herbal medicine in China and other Asian countries since ancient times  and Lactobacillus gasseri B1-27 (Genome Accession Number NR113820.1) strains were inoculated into the medium at a volume of 200 μl and placed in an incubator set to 37 ± 1°C for 24 h for anaerobic culture. After confirming the absence of infectious microorganisms using an optical microscope, the broth was activated three times using 2% inoculum to keep the bacteria at high viability for future use.The MRS broth was prepared, sterilized at 121°C for 20 min, and then dispensed into 10 ml sterilized test tubes in a BioClean room. The preserved Poria cocos powder . After stirring a blank control fermentation broth and four Poria cocos fermentation broths at 1, 2.5, 5 and 7.5% were obtained. Subsequently, 10 ml of culture medium in each conical flask was dispensed into five test tubes. After sterilization at 121°C for 20 min, 200 μl of culture medium was separately inoculated with L. plantarum R9 and L. gasseri B1-27 for culture.First, 250 ml of MRS broth liquid medium was prepared. After adjusting its pH to 6.25, the broth was then evenly dispensed into five conical flasks and supplemented with 0, 0.5, 1.25, 2.5, or 3.75 g of Poria cocos at 0, 20, and 24 h after inoculation. Each measurement was repeated three times.A spectrophotometer  and a pH meter  Co., Ltd., Shanghai, China) were used to measure the absorbance  at 600 nm and the pH value of the fermentation broths with different concentrations of Poria cocos fermentation broth was calculated throughout 0–20 h after the onset of the fermentation, using the formula E = ΔODexperimental group − ΔODcontrol group. When E > 0, the Poria cocos fermentation broth was considered to have a proliferation-enhancing effect on the probiotics derived from human milk, whereas when E ≤ 0, there was no proliferation-enhancing effect , filtered through a 0.22 μm membrane, and transferred into a detection bottle for liquid chromatography-mass spectrometry (LC–MS) analysis  column . The temperature of the column was maintained at 40°C. The flow rate and injection volume were set to 0.25 ml/min and 2 μl, respectively. For LC-electrospray ionization (ESI)(+)-MS analysis, the mobile phases consisted of (C) 0.1% formic acid in acetonitrile (v/v) and (D) 0.1% formic acid in water (v/v). Separation was conducted using the following gradient: 0–1 min, 2% C; 1–9 min, 2–50% C; 9–12 min, 50–98% C; 12–13.5 min, 98% C; 13.5–14 min, 98–2% C; 14–20 min, 2% C. For LC-ESI (−)-MS analysis, the analytes were separated with (A) acetonitrile and (B) ammonium formate (5 mM) using the following gradient: 0–1 min, 2% A; 1–9 min, 2–50% A; 9–12 min, 50–98% A; 12–13.5 min, 98% A; 13.5–14 min, 98–2% A; 14–17 min, 2% A  equipped withthe ACQUITY UPLCin, 2% A .MS detection of metabolites was conducted on a Q Exactive Focus instrument  using an ESI ion source. Simultaneous MS1 and tandem MS/MS  acquisition was used. The parameters were as follows: sheath gas pressure, 30 arb; aux gas flow, 10 arb; spray voltage, 3.50 kV and − 2.50 kV for ESI(+) and ESI(−), respectively; capillary temperature, 325°C; MS1 range, m/z 81–1,000; MS1 resolving power, 70,000 FWHM; the number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM; normalized collision energy, 30%; dynamic exclusion time, automatic .Proteowizard software package  were applied to determine the variables that contributed to metabolite classification. Finally, p < 0.05 and VIP values >1 were considered statistically significant. The MetaboAnalyst software package  and orthogonal partial least squares discriminant analysis (OPLS-DA) in the R software package ple data . The met package  was usedPoria cocos fermentation broth had the strongest proliferation- enhancing effect on L. plantarum R9 and L. gasseri B1-27 (L. plantarum R9 (p = 0.001) and L. gasseri B1-27 in the 2.5% Poria cocos fermentation broth were significantly increased after 20 h of fermentation (p = 0.013). In addition, 7.5% Poria cocos powder inhibited the growth of both probiotics  benzoic acid. A total of 35 and 45 differential metabolites were detected in L. plantarum R9 and L. gasseri B1-27, including organic acids and their substituted derivatives, amino acids and their derivatives, flavonoids, vitamins, purines, and pyrimidines and their derivatives were identified. The differential abundance of these metabolites is displayed as a heatmap in Poria cocos were significantly different before and after fermentation. Generally, the content of metabolites in the Poria cocos fermentation broth after L. gasseri B1-27 and L. plantarum R9 fermentation was lower than that before fermentation, with similar metabolic patterns within each group. The content of organic acids and their substituted derivatives, as well as amino acids and their derivatives, were higher after fermentation than that before fermentation  were analyzed. Usually, the concentration of fermentation additives is optimal at 1–10%, and excessive addition may affect the organoleptic properties of fermented products. By comparing the OD value of the two probiotics in Poria cocos fermentation broth at the tested concentrations 20 h after the onset of fermentation  are a large and ancient family of transmembrane transport proteins, which are involved in physiological activities such as the transport and accumulation of metabolites, detoxification of harmful substances, stomatal regulation, and plant defense  and the Beijing Innovation Team of Livestock Industry Technology System, Daxing District Major Scientific and Technological Achievements Transformation Project (grant no. 2020006).Authors QW, KY, XW, WQ, and LC were employed by Beijing Sanyuan Foods Co. Ltd.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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