Chronic myelomonocytic leukemia (CMML) is a hematologic disorder that is an overlap syndrome between myelodysplastic syndromes and myeloproliferative neoplasms, and can be associated with autoimmune and inflammatory diseases. This study aimed to describe kidney involvement in patients with CMML, their treatments, and outcomes.We conducted a French and American multicenter retrospective study in 15 centers, identifying patients with CMML with acute kidney injury (AKI), chronic kidney disease (CKD), and urine abnormalities.n = 14; median age 76.5 years [71.9–83]) developed a kidney disease 6 months [1.6–25.6] after the diagnosis of CMML. At the time of kidney disease diagnosis, median urinary protein-to-creatinine ratio was 2 g/g [1.25–3.4], and median serum creatinine was 2.26 mg/dl [1.46–2.68]. Fourteen patients (87.5%) underwent a kidney biopsy, and the 2 main pathological findings were lysozyme nephropathy (56%) and renal infiltration by the CMML (37.5%). Ten patients received a new treatment following the CMML-associated kidney injury. Among patients with monitored kidney function, and after a median follow-up of 15 months [9.9–34.9], 4 patients had CKD stage 3, 4 had CKD stage 4, 1 had an end-stage kidney disease. In our patient series, 2 patients evolved to an acute myeloid leukemia (AML), and 5 died. Compared with 116 CMML controls, patients who had a kidney involvement had a higher monocyte count (P < 0.001), had more CMML-1 (P = 0.005), were more susceptible to develop an AML (P = 0.02), and were more eligible to receive a specific hematologic treatment, with hydroxyurea, or hypomethylating agents (P < 0.001), but no survival difference was seen between the 2 groups (P = 0.6978).Sixteen patients (males, In this cohort of patients with CMML with a kidney injury, the 2 most frequent renal complications were lysozyme-induced nephropathy and renal infiltration by the CMML. Kidney involvement should be closely monitored in patients with CMML.  Kidney diseases in patients with hematological malignancies are common, with various etiologies.CMML is an overlap syndrome that has characteristics of a myelodysplastic syndrome, whereas other findings are more consistent with the diagnosis of myeloproliferative neoplasm.,CMML can be associated with systemic inflammatory and autoimmune diseases in 10% to 30% of patients, mostly neutrophilic diseases, connective tissue disorders, arthritis, and vasculitis.The objective of this work was to study the clinical and histological presentation of patients with renal impairment during CMML, as well as their therapeutic management in a multicenter cohort. Then, we compared the hematological phenotype of patients with a kidney injury occurring during CMML with CMML patients’ controls without kidney disease.Patients with an established diagnosis of CMML and diagnosed with AKI, CKD, or urine abnormalities were retrospectively included. We collected data of patients from 3 different sources . The firDiagnoses criteria for CMML were based on the World Health Organization classification revised in 2016. CMML were classified as CMML-0 , CMML-1 , and CMML-2 . Cytogenetic analysis and molecular findings were collected when available in the center.2), or markers of kidney damage: albuminuria with albumin-to-creatinine ratio ≥30mg/g, urine sediment abnormalities, electrolyte and other abnormalities due to tubular disorders, and abnormalities detected by histology among other things.We included adult patients (>18 years old) diagnosed with CMML, and a kidney disease less than 10 years apart. According to the National Kidney Foundation’s Kidney Disease Outcome and Quality Initiative guidelines, CKD is defined as either a decreased glomerular filtration rate  for the binary variables, and in median (interquartile range) for the continuous variables. Given the size of the numbers and the lack of a normal distribution of values, we used nonparametric tests. Comparisons of continuous variables between 2 groups were made using the Mann-Whitney test, and those of binary variables were made using the Chi-square test. A Kaplan-Meier method was used to estimate overall survival. The significance level was Statistical analyses were performed using GraphPad Prism v8.00 for MacOS , and R v4.0.3 for MacOS .This study was conducted according to the Declaration of Helsinki and fulfilled the recommendations of the French “Commission Nationale Informatique & Libertés” CNIL. According to its policy, this study was approved by the Institutional Review Board of the Brigham and Women’s Hospital (protocol number 2019P003787), who validated a waiver of consent.n = 14, aged 76.5 years [71.9–83]) with a kidney disease associated with CMML were included in this study. CMML disease subtypes were as following: type 0 in 5 patients (25%), type 1 in 10 patients (66.7%), and type 2 in 1 patient (8.3%) , PTPN1, NRAS in patient FRA-02; JAK2 in patient FRA-04; NRAS, KRAS, ASXLI1 in patient FRA-11; EVI1 duplication in patient FRA-28; and BCORL1, NRAS, PHF6, SRSF2, TET2, ZRSR2 in patient FRA-32.Overall, 16 patients  . Median 2. Median proteinuria was 2 g/g (1.25–3.4), with a microscopic hematuria present in 6 of the 10 patients with an evaluation of the urine sediment. Leukocyturia was available in 8 patients with a positivity in 25% of the cases.Time between the hematologic and kidney disease was 6 months [1.6–25.6] . The dian = 9; 64%), then nephrotic syndrome , rapidly progressive glomerulonephritis , and a progressive CKD . A preexisting CKD of unknown cause with an eGFR of 33 and 44 ml/min per 1.73 m2 was present in 2 patients. Hypertension was present in 82% of the patients, whereas diabetes represented 18%. Few patients had systemic symptoms  with 2 organs preferentially incriminated: skin and lungs . Four patients had a monoclonal immunoglobulin (3 IgG kappa and 1 IgG lambda) with a median peak of 3 g/l [1.6–9]. Median C-reactive protein was 56 mg/l [9.6–100]. Auto-immunity was detected in 5 of the 10 tested patients . One patient had a detectable cryoglobulinemia associated with a diagnosis of lysozyme nephropathy. Regarding the serum complement analyses, 25% had a low C3 and 12.5% a low C4.The first indication of the kidney biopsy was AKI associated to a tubular proteinuria , and an interstitial infiltrate compatible with a CMML infiltration in the kidney   underwent a kidney biopsy. The 2 most frequent diagnoses were lysozyme nephropathy  . Among tn = 4), and a vacuolization of the epithelial cells of the proximal tubules (n = 7). Lysozyme staining was positive in 5 (n = 62.5%) patients (4 immunohistochemistry and 1 immunofluorescence). Four patients with lysozyme nephropathy had also lysozymuria. Interstitial fibrosis ranged from 10% to 30%. Inflammatory infiltrate with mostly mononuclear myeloid cells was described in 9 patients. For the patients who had a characterization of the infiltrate by immunohistochemistry, MPO and CD68 staining were positive  for 5 (45.5%) patients and got improved in 5 (45.5%) patients (4 missing data). One patient refused to be treated by azacitidine, and his kidney function remained stable after 68 months.After a median follow-up of 15 months (9.9–34.9), hematologic malignancy worsened in 6 patients , with 5 deaths at last follow-up . After treatment, kidney function worsened in 1 (9%) patient. It was stable (eGFR change ± 5 ml/min per 1.73 mP < 0.001) (P = 0.005). Even if there was no difference in terms of prognostics, with an International Prognosis Scoring System-Revised above 3.5 in 28.4% of CMML control group against 20% (P = 1), patients with CMML with a kidney injury were more susceptible to develop an AML: 30.8% versus 6.9% in the control group (P = 0.02). Patients with CMML who had a kidney injury were more eligible to receive a specific hematologic treatment, with hydroxyurea, or hypomethylating agents . The median follow-up from CMML diagnosis was 21 months [7–39.5] in patients without a kidney injury, and 34.9 months [13.9–46.5] in patients with a kidney injury (P = 0.174). The median survival was 59 months, and not reached for the follow-up period in CMML with and without a kidney injury, respectively   . Moreove3–3.28]) .Table 3CCMML is a myeloid neoplasm characterized as a myelodysplastic syndrome/ myeloproliferative neoplasm overlap syndrome. CMML is associated with significant monocytosis, which is correlated with AKI and CKD.et al.et al.et al.,In our study, the time between the diagnosis of CMML and the development of a kidney injury was 6 months, which is close to the delay reported by Grignano Lysozyme is a small cationic protein produced by monocytes, that is freely filtered by the glomerular filtration barrier, and reabsorbed by proximal tubules.,,,Kidney infiltration by CMML is mostly seen in the tubulointerstitial compartment, even if it has already been described in the glomerulus.et al.et al.At last follow-up, kidney function evolution was heterogenous, with 4 patients evolving to CKD 3, 4 patients to CKD 4, and 1 patient to end-stage kidney disease, which was comparable to Belliere Limitations of this work are mainly due to its retrospective design, and the small sample size of the cohort. To push further the analysis, the next objectives will be to collect kidney biopsy samples to further characterize the pathological features of the kidney injuries (infiltrate and lysozyme staining) associated with CMML. One of our perspectives will be to realize next-generation sequencing on the kidney biopsies of patients with an infiltrate, to see if we can find a significant renal clonal infiltrate of CMML cells.In conclusion, a regular screening for blood pressure, proteinuria, and kidney function should be proposed to all patients with CMML. In case of kidney dysfunction, kidney biopsy seems important to confirm the diagnosis in patients with CMML or to rule out differential diagnoses. Dosage of lysozymuria can also be informative, although not easily accessible in routine. In this cohort of patients with CMML with a kidney injury, the 2 most frequent kidney complications were lysozyme-induced nephropathy, and kidney infiltration by the CMML. The occurrence of a kidney injury in the course of a CMML seems to worsen the prognosis of these patients.List of the MINHEMON group: Marie-Camille Lafargue, Mickaël Bobot, Thibault Comont, Nathalie Cambier, Kamel Laribi, Olivier Fain,Lionel Adès, Pierre Fenaux, Arsène MekinianList of GFM group: Thibault Comont, Nathalie Cambier, Kamel Laribi, Olivier Fain, Lionel Adès, Pierre Fenaux, Arsène MekinianList of French VEXAS group: Thibault Comont, Olivier Fain, Lionel Adès, Pierre Fenaux, Arsène MekinianAM is investigator of CELGENE, ROCHE, CHUGAI founded trials with APHP and Hopital 15-20 promotion; AM received several fees for congress travels and experts’ use from LFB, SANOFI, SHIRE, and CELGENE. All other authors have declared no conflicting interest.


A. tumefaciens (GV3101) harboring the pMDC32 vector containing hpt or the GmUbi-35S-GUS vector containing the GUS gene during two separate events. High transient GUS expression was obtained during cotyledon explant culture on MS media supplemented with 2.5 mg/L aztreonam. The aztreonam-treated explants showed high efficiency in transient and stable transformation as compared to the untreated control. The transformation of aztreonam-treated explants during seed imbibition resulted in an average of 21.1% as compared to 13.2% in control by using the pMDC32 vector and 28.5 and 20.7% while using the GUS gene cassette, respectively. Based on these findings, the metabolic analysis of the explant after aztreonam treatment was assessed. The high accumulation of flavonoids was identified during an untargeted metabolic analysis. The quantification results showed a significantly high accumulation of the four compounds, i.e., genistein, apigenin, naringenin, and genistin, in cotyledon explants after 18 hours of aztreonam treatment. Alongside this, aztreonam also had some surprising effects on root elongation and lateral root formation when compared to indole-3-butyric acid (IBA). Our findings were limited to soybeans. However, the discovery of aztreonam and its effect on triggering flavonoids could lead to the potential role of aztreonam in the agrobacterium-mediated transformation of different crops.Agrobacterium-mediated soybean transformation is the simplest method of gene transfer. However, the low transformation due to the intractable nature of soybean genotypes hinders this process. The use of biochemicals  plays an important role in increasing soybean transformation. These biochemicals induce chemotaxis and virulence gene activation during the infection process. Here we identified a biochemical, aztreonam (a monobactam), for high agrobacterium-mediated transformation in soybean. The soybean explants from three genotypes were inoculated with  A binary vectors, pMDC32, and GmUbi-3XFlag-35S-GUS/GFP were used to transfer US genes . The pMDin silico vector assembly was performed in Snapgene software. The GUS gene was cloned into the AscI and XbaI restriction sites of the GmUbi-3XFlag-GFP vector. We additionally employed the CaMV-35S promoter in place of the GmUbi promoter for stable GUS expression. Restriction of the vector was carried out in a 50 mL aqueous reaction volume containing 1 μL AscI and 1 μL XbaI endonucleases (Takara), 1X Quick cut buffer (Takara), and 1 μg vector. The reaction mixture was incubated at 35°C for 2 h. The GUS gene was amplified from the pCambi 1,300 vector by using a pair of overlapping primers (A. tumefaciens (GV3101) after colony PCR and sequence confirmation.For the cloning of the GUS gene in the GmUbi-3XFlag vector,  primers . Ligatio2.2.2.6H2O 0.02 mg/L, agar 0.7%, sucrose 1%, and aztreonam 2.5 mg/L, pH 5.7, as treatment and control without aztreonam. The seeds were kept in a growth chamber for 18 h (in the dark) at a constant temperature of 25°C and 70% relative humidity. During each event, the Agrobacterium containing the pMDC32 or GmUbi-3xFLAG-GUS assembly vectors was transformed into a soybean cotyledonary node. Prior to inoculation, A. tumefaciens was incubated overnight in YEP media containing selective antibiotics, as described by Agrobacterium culture was centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the pellet was re-suspended in inoculation medium containing half-strength MS media with vitamins and 30 g/L sucrose, pH 5.4. The medium’s temperature was maintained at 21°C, and the OD was adjusted to 0.7 ± 0.02 at 600 nm. Following OD adjustment, 250 mg/L of L-cysteine and 200 μM acetosyringone were added to the inoculation medium. Before infiltration into the cotyledons, the inoculation medium was incubated at room temperature for 30 min. Imbibed seeds were divided into halves with care to ensure each cotyledon received a portion of the embryonic axis. The plumules were gently cut off. Multiple cuts were made adjacent to the cotyledonary node. According to For half-cotyledonary node transformation and direct shoot organogenesis, freshly harvested soybean seeds from three soybean genotypes  were employed. The seeds were sterilized in 70% ethanol for 5 min and then in a 4.5% sodium hypochlorite solution for 15 min. After sterilization, the seeds were rinsed three times with ddH2O. The sterilized seeds were placed on germination medium containing quarter-strength MS medium with B5 vitamins, MgCl2.3.3 0.3 mg/L, and aztreonam 2.5 mg/L (only in the case to treat explants) pH 5.4). However, the control media omitted aztreonam. The parafilms were wrapped tightly around the co-cultivation plates, which were then left in the dark for 4 days (under the aforementioned germination conditions). In order to eradicate the adhering Agrobacterium, the cotyledons were briefly washed in washing medium (as indicated for co-cultivation supplemented with 250 mg/L carbenicillin and 250 mg/L cefotaxime) for 30 min. Following this, the explants were transferred to shoot induction media.The half-cotyledonary explants were co-cultivated in 14 cm culture plates. Adaxially facing down, treated cotyledons were placed on sterile filter paper. The filter paper was soaked with liquid co-cultivation media , spermidine 2.5 mg/L (Solarbio cat# S8030), N6-(2-isopentenyl) adenine 2iP 0.4 mg/L (cat# B24576), kinetin 0.3 mg/L , GA3 0.3 mg/L (Solarbio cat# G8910), cefotaxime 150 mg/L, carbenicillin 200 mg/L (Solarbio), 2-(N-Morpholino) ethane sulfonic acid monohydrate (MES)  1.5 g/L, pH 5.7). Cotyledonary explants were placed 45 degrees apart horizontally in SIM. The explants were cultured in a growth chamber under fully controlled conditions  for 16 h of light and 8 h of darkness. After 14 days in each sub-culture, the explants were transferred to new SIM media. When necessary for the aforementioned various experiments, elongating shoots (2–3 cm) were cut off from the cotyledon explant after 14 days. The excised shoots were rooted on root induction media containing half-strength MS with B5 vitamins, 2% sucrose, 0.75 mg/L MgCl2. 6H2O, 1.5 g/L MES, 4 mg/L IBA, 150 mg/L cefotaxime, and 180 mg/L carbenicillin, pH 5.7. Instead of IBA, some explants were planted on RIM supplemented with 2.5 mg/L aztreonam. The rooted seedlings were transferred to 6 cm2 pots containing a vermiculite:peat (1:1) mix. The trays containing pots were covered with transparent plastic humidity domes. After 7 days of acclimatization, the seedlings were transferred to the greenhouse in large (14 cm) pots.For shoot organogenesis, we used our newly optimized SIM . PCR was conducted for the identification of the hpt gene from soybean lines with a pair of hpt-specific primers. The forward and reverse primer sequences were hpt-forward (ATTTGTGTACGCCCGACAGT) and hpt-reverse (CTCTCGGAGGGCGAAGAATC). PCR cycle conditions were 94°C for 4 min, 94°C for 30 s, 51.3°C for 30 s, 72°C for 30 s (34 cycles), 72°C final extension for 5 min, and then 4°C hold. However, for T1 transgene identification, the number of PCR cycles was raised to 40. The PCR products were resolved on a 1.5% agarose gel. The vector and non-transformed soybean lines were used as positive and negative controls in the gel, respectively. The putative transgenic soybean lines’ positive bands at a length of 840 bp were identified as hpt gene bands. Following the directions in the user’s manual, the Omega Bio-TEK (D2500-02) gel extraction kit was used to extract the DNA bands from the agarose gel. Sequencing provided further assurance of the targeted band. At maturity, seeds from positive transgenic T0 plants were harvested. In the T1 generation, segregation in transgenic events was evaluated. Similar procedures were employed for the identification of transgenic lines, as mentioned before.In the greenhouse, the fully acclimatized seedlings were allowed to grow and mature. With some modest modifications, the CTAB technique was used to extract the total genomic DNA from soybean lines . The PCR2.6.Soybean explants from three genotypes were subjected to GUS histochemical analysis. After 4 days of co-cultivation, GUS expression was examined in the inoculated epicotyl sections. Regenerated shoots were then tested 14 days later. The GUS staining buffer (50 mM NaH2PO4 (pH 7.2), 10 mM Na2EDTA, 0.1% (v/v) Triton-X, 1 mM K4Fe(CN)6, 1 mM K3Fe(CN)6, and 2 mM X-gluc) was used to stain the inoculated cotyledon parts . The cot2.7.To determine the effect of aztreonam on soybean root. The newly excised explants were planted in 1/2 MS media with 1% sucrose, 0.7% agar, 100 mg/L carbenicillin, and 100 mg/L cefotaxime, with 2.5 mg/L aztreonam (treatment) and IBA as a control. Aztreonam and IBA were added after the media had been autoclaved. Aztreonam’s performance was assessed in a comparative study of root induction and elongation using indole-3-butyric acid (IBA) as a control. More than six explants were cultured in each RIM medium pot and allowed to root. After 7 days of culture, root length and the number of lateral roots were evaluated in treated and control explants.2.8.We conducted an untargeted metabolic study on soybean explants to find out the metabolic modulation following aztreonam application. The soybean seeds were cultured on solid MS medium containing 2.5 mg/L aztreonam (treated) and without aztreonam (control). Six seeds in each pot were given 18 h of regulated soaking at 25 degrees Celsius, 70% humidity, a 16/8 light/dark cycle, and 60% white florescence light. The epicotyl and cotyledon portions were collected in separate tubes. The cotyledons were washed with tap water to remove adherent MS media. The samples were quickly frozen in liquid nitrogen before being crushed into a fine powder. From each sample, 100 mg of powder was collected in 5 mL polyethylene tubes. The powder was dissolved in 3 mL of 80% methanol. The samples were homogenized  for 10 min at 4000 rpm. Centrifugation of the homogenate was performed at 4000 rpm for 15 min. The supernatant from each sample was collected in a fresh 5 mL tube. The solution was desiccated (in an Eppendorf centrifuge and concentrator) for 6 h at 30°C. The dried masses were redissolved in 300 μL of an 80% cold methanol and 0.01% formic acid (v/v) solution. Homogenate was filtered using a syringe filter  before filling an HPLC vial. Each HPLC vial was filled with 200 μL. To identify contaminants or analytical errors during sample processing, a field blank sample was employed. For quality control, an even volume of each sample was taken and mixed in a single HPLC vial. Chrysin (1 μL/mL) was used as an internal standard. The experiment was repeated three times with three biological replicates.For the extraction of flavonoid compounds, 96% (v/v) ethanol was used. From the laboratory chemicals repository, five HPLC-grade standards  were obtained. The compounds were dissolved in DMSO to create a standard solution for measuring flavonoids in soybean tissue. In order to generate calibration curves, standards were further diluted and run through a UPLC-QqQ-MS/MS apparatus.2.9.The metabolic analysis was conducted using two separate UHPLC systems. For an untargeted metabolic investigation, the samples, a control, and a blank were placed into the LC–MS Q-Executive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific United States). The primary assembly was equipped with an auto-detector, auto-sampler, and pump that were all regularly monitored. The C18 column  was used for the separation of metabolites. Flavonoids were quantified using the ultra-high performance liquid chromatography triple quadrupole mass spectrometry technique (UHPLC-QqQ-MS/MS) (Acquity UPLC I-Class Plus). A binary solvent phase was used as an elution gradient system for LC–MS/MS. These solvents contained 0.1% formic acid/water (solvent A) and methanol (solvent B). Both solvents were degassed in a Branson Ultrasonic Cleaner USA (Model CPX5800H-C) for 10 min before use. The elution gradient was used as follows: 0 min 10% B; 2 min 10% B; 10 min 50% B; 10 min 80% B; 13 min 95% B; 14 min 95% B; 14 min 10% B; and 18 min 10% B. The total run time ranged from 0 to 18 min. The flow rate was 0.3 mL/min, and the sample injection volume was 10 μL. The wavelength ranged from 190 to 500 nm. The sheath gas flow rate of 35, the auxiliary gas flow rate of 15, and the sweep gas flow rate of 0 were maintained. Spray voltage and current were maintained at 3.21 kV and 16.40 angstrom, respectively. The auxiliary gas heater temperature was 348°C, while the capillary temperature was 320°C. The spectra were acquired in negative and positive ionization modes over a mass-to-charge (m/z) ratio ranging from 50 to 1,200. The data was collected at a rate of 0.2–250 Hz. The scan range for both positive and negative ionization modes was 70 to 1,050 m/z, with a full MS resolution of 70,000.The binary solvents chose in the elution gradient technique for quantitative determination of flavonoids were (A) water/formic acid (0.01%) and (B) 80% methanol/0.01% formic acid. A stationary-phase Acquity UPLC Cortecs T3 column  was used. For the elution gradient, 0.4 mL/min flow was maintained. The elution gradient was set as 0 min 5% B, 0.5 min 5% B, 2.5 min 20% B, 13 min 80% B, 15 min 95% B, 16 min 95% B, and 18 min 5% B. All the other conditions were the same as mentioned above.2.10.The accurate m/z value that was passed down from ESI-Q-TRAP LC–MS/MS was analyzed further. Fragmentation pattern and retention time were processed in thermo-scientific proteome discoverer software. The metabolite identification and structural similarities were retrieved by following the best match percentage rules, m/z fragmentation pattern, and least molecular weight differences<0.01. The compounds with m/z fragments that matched ≥70% with reference libraries were kept for further analysis. To remove the ambiguity, some identified compounds were further verified by the PubChem2.11.p < 0.05. A post-hoc analysis of Fisher’s least significant difference was generated using the Pearson distance measure and the Ward clustering algorithm. For the assessment of variation among root length and number of lateral roots, an unpaired T-test was applied in GraphPad Prism 8 and MS Excel 2016. The pathway analysis and metabolite enrichment analysis were performed on the MetaboAnalyst platform by comparing sorted metabolites with Arabidopsis metabolite libraries. For graphics and figure illustrations, CorelDraw Graphic Suite 2021 was used. The Snapgene software (4.1.9 version) was used for computer simulations of vector assembly and gene cloning. All the experiments were repeated at least three times with three or more biological replicates.Multiple-variate analysis of untargeted metabolites was performed using the web interface of the MetaboAnalyst V 5.0 companion with the R package . The acquired data was normalized prior to analysis. For the analysis of statistical significance among groups, a one-way ANOVA was used. Multivariate strategies for comprehensive data analysis, like a supervised method  and an unsupervised method , were used. Differentially accumulated compounds were identified through PLS-DA and a variable importance in projection (VIP) score using a significance threshold of 3.3.1.Agrobacterium were investigated at such a low concentration. The cotyledonary explants without aztreonam were kept as controls. When compared to the control, the elongated epicotyl segment of the aztreonam-treated explants showed stronger signals and a greater number of positive GUS-stained tissues  (p = 0.002) when treated with either aztreonam or IBA. Aztreonam generated long white shoots, but root growth was slow in the IBA-containing medium. Visual examination revealed differences in root pattern, such as root color and root thickness  . The roohickness . Accordi3.3.p-values<0.05  were quantified in aztreonam-treated and control explants. The ethanolic extracts of the aztreonam-treated and control cotyledon explants were quantified using UHPLC-QqQ-MS/MS. The precise quantification of each compound was acquired by comparing it to known concentrations of that compound. During the quantification, one additional peak was identified as genistin, which was abundant in the treated samples. Statistical analysis showed that the accumulation of four compounds, i.e., genistein (p = 0.000007), apigenin (p = 0.0061), naringenin (p = 0.0015), and genistin (p = 0.0016), was significantly higher in cotyledon explants after aztreonam treatment  gene-inducing chemicals, medium composition, and tissue specific factors are critical for boosting transformation efficiency  can be found at this link: MW: Conceptualization, Formal analysis, Methodology, Software, Writing – original draft. WY: Methodology, Writing – review & editing. KY: Writing – review & editing. XZ: Project administration, Supervision, Writing – review & editing.The author(s) declare financial support was received for the research, authorship, and/or publication of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rhodopseudomonas palustris strains of VNW02, TLS06, VNW64, and VNS89, mixed with spent rice straw (SRS) from mushroom cultivation as a carrier on promoting sesame growth and yield, and ameliorating the alluvial soil (AS) fertility in dykes. A 4 × 3 factorial experiment consisting of different levels of the solid PNSB biofertilizer mixture at 0, 3, 4, and 5 t·ha−1 , and nitrogen (N) and phosphorus (P) inorganic fertilizer rates  was performed in pots with the sesame variety of ADB1 in the dyked AS. The solid PNSB biofertilizer mixture at at least 3 t·ha−1 significantly enhanced the sesame seed yield by providing higher macronutrients for plants by increasing available N and soluble P concentrations in the soil. The solid PNSB biofertilizer mixture in addition to 75% of the recommended N and P fertilizers produced an equivalent yield in comparison to the utilized 100% of N and P inorganic fertilizers. The solid PNSB biofertilizer mixture in the SRS from the mushroom production reduced at least 25% of N and P chemical fertilizers for gaining the maximal seed yield and enriched soil characteristics for the sustainable black sesame cultivation in the dyked AS.The aim of the current research was to evaluate the effects of members of purple nonsulfur bacteria (PNSB),  It c in 2018 .Alluvial soil (AS) is a soil that is deposited by the surface water and can be found along rivers and their banks, in floodplains and deltas. This leads to high nutrient concentrations within the AS; thereby, the soil in An Giang province has been intensively used for crops that lack available nutrients from their indigenous soil . HoweverThe use of fertilizers for sesame is a notorious concern in most aspects in developing countries , particuRhodopseudomonas spp. as biofertilizers in paddy fields have been reported to improve adverse effects of soil environments such as acidity, salinity, and toxicity [Stevia rebaudiana [Purple nonsulfur bacteria (PNSB) are N-fixers with ability to grow in different growth modes as a photoautotroph which can make use of light and carbon dioxide in the environment to produce its own energy via the process of photosynthesis, photoheterotrophs, chemoheterotrophs, and chemoautotrophs depending on the environmental condition –22. Thistoxicity –29 to entoxicity , 30–33 abaudiana , cucumbebaudiana , 36, andbaudiana . RegardiRhodopseudomonas strains, particularly R. palustris VNW02, TLS06, VNW64, and VNS89 strains which were isolated from various acid sulfate soils [3+ and Fe2+ [R. palustris in SRS from mushroom cultivation as a carrier to support sesame growth and yield, along with improving the AS fertility in dykes, for the possibility to achieve an alternative approach to reduce chemical fertilizers for a sustainable sesame cultivation.Interestingly, the biofertilizers of acidic-resistantte soils  can act and Fe2+ . Fortunaand Fe2+ , 33 and and Fe2+  to suppoThe soil used in the current study was collected at 0–20 cm depth from a previously sesame cultivated soil in Chau Phu district, An Giang province, Viet Nam. Results of the initial soil physiochemical parameters, which were determined following the method in R. palustris, VNW02, TLS06, VNW64, and VNS89, were used as a mixed biofertilizer in this study, and their accession numbers from the National Center for Biotechnology Information (NCBI) were KY624605, KY624609, KY624606, and KY624607, respectively [The four promising strains of ectively .The SRS originating from mushroom cultivation was used as a carrier for the mixed biofertilizer.The black sesame variety ADB1, which is commonly planted in the Mekong Delta, was collected and kept in the Crop Science Faculty, College of Agriculture, Can Tho University.Each plastic pot, whose size was 25 and 21 cm in diameter at the top and bottom and 21 cm in height, was used and had seven holes at the bottom.2O5), and potassium chloride for the K application (60% KCl).The actual chemical fertilizers used were urea for the N application (46% N), superphosphate fertilizer for the P application (16% P(1) PNSB Preparation. The four PNSB strains were activated in the basic isolation medium (BIM) broth for 48 h under microaerobic light conditions. After that, the optical density of each culture was adjusted to 1.2 at the wavelength of 660 nm by diluting in sterile distilled water, with the final density of 109 cells·mL−1. Each cell suspension was ready to be used as an inoculant.(2) Sesame Seed Preparation and Bacterization. The black sesame seeds were first sanitized by submergence in 70% ethanol for 3 min and 1% sodium hypochlorite for 10 min, and then washed twice with sterile distilled water. The black sesame seeds were incubated for 24 h under dark conditions for germination. Subsequently, for the seed bacterization, 1,000 sesame seeds were mixed well with a 10 mL bacterial cell suspension of 109 cells·mL−1 (2.5 mL for each strain) for 1 h in a reciprocal shaker at 60 rpm. After 2 h of mixing, the seeds were taken out and dried under the laminar air flow before planting. The control seeds were similarly prepared, but in sterile distilled water instead of the bacterial suspension. Five seeds were grown in a pot, so the density of the mixed 4 PNSB was roughly 0.5 × 108 cells pot−1.(3) Preparation of the Mixed PNSB Biofertilizer in a Solid Form. The SRS from mushroom cultivation was sterilized at 121°C for 30 min and was used as a carrier for the PNSB biofertilizer based on its low nutrient content and poor decomposition. Then, 10 mL of 4 PNSB inoculants (2.5 × 4 each) was added to 100 g of the carrier and incubated for one week before use. This product was named as the solid mixed PNSB biofertilizer containing roughly 108 cells g−1 and is called as solid PNSB biofertilizer in short.(4) Application of the Solid Mixed PNSB Biofertilizer. A 50% of the designated weight of the solid PNSB biofertilizer was applied on the soil surface at 0 and 20 days after sowing. It was put near sesame roots with designated levels. The solid PNSB biofertilizer consisted of 2.37% N, 1.76% P, 1.20% K, and C/N ratio (20.0); the PNSB density was 0.87 × 107 cells·g−1. Thus, the total PNSB density in each pot was 1.81 × 108 cells pot−1 that corresponded to 3 t·ha−1, 2.24 × 108 cells pot−1 for 4 t·ha−1, and 2.68 × 108 cells pot−1 for 5 t·ha−1. The methods were described by Sparks et al. [s et al. . In deta(1) Soil Preparation. RS residues were removed from the used soil before mixing to avoid its effects on the soil fertility and drying in the ambient atmosphere. Then, 10 kg of soil in each pot was moistened with roughly 1 L pot−1 of tap water for 2 days before sowing.(2) Chemical Fertilizer Formula. N, P, and K fertilizers were used as the recommendation formula of 100N-60P2O5-30K2O. Each treatment was applied as a designated volume. The fertilization was divided into 4 times. For the first time, 100% phosphorus was applied as a base. In the second time, 10 days after sowing (DAS), 30% N and 50% K were applied. In the third time, 40% N was applied at 20 DAS. Finally, 30% N and 50% K were applied [(3) Sesame Planting. Each pot contained 10 kg of dry soil with five plants (one plant in the center and four plants at four corners).(1) Initial and Harvest Soil Property. The methods for the soil analysis are described as follows: Soil chemical parameters in KCl) or deionized water (pHH2O) at a ratio of 1 : 5 for soil : solvent. The total iron was determined by using the ICP-OES. The Alexchangeable concentration was extracted by 1 M KCl, and the spectrophotometric method was employed for the aluminum ion quantization from the soil supernatant by the fluorometric analysis of the Al3+ content with 8-hydroxyquinoline and butyl acetate. The Feactive concentration in soil samples was extracted by diethylenetriaminepentaacetic acid (DTPA). The digested solution was used for the amorphous iron oxide analysis, which was analyzed by ammonium oxalate acid in the dark and detected by the modified Tamm's reagent. The ferrozine method was described for determination of the Fe2+ concentration via the 1,10-phenanthroline reagent using a visible spectrophotometer at the wavelength of 562 nm. The available P was determined by the Bray II methods, and fractions of inorganic P were extracted by 0.5 M NH4F, 0.1 M NaOH, and 0.25 M H2SO4 for aluminum-phosphorus (Al-P), iron-phosphorus (Fe-P), and calcium-phosphorus (Ca-P), respectively. The total N was quantified by the regular Kjeldahl method via destruction of organic N prior to the inorganic N determination. The NH4+−N concentration was distillated by a titrimetric procedure. The dichromate oxidation procedure by the thermal conductivity technique of sulfuric acid was utilized for converting to the inorganic carbon from organic carbon compounds prior to titrate with ferrous sulfate heptahydrate for the total carbon determination. The cation exchange capacity (CEC): A compulsive exchange method has been recommended for the determination of CEC in the soil. This extracted solution was also used for the analysis of potassium, sodium, calcium, and magnesium by an atomic absorption spectrophotometry.(2) PNSB Population. The density of the PNSB from the harvest soil was enumerated by the most probable number technique as described by Harada et al. [a et al.  and modia et al. .−1 and the chemical fertilizer with three levels of 50, 75, and 100% of N and P of the recommendation formula of chemical fertilizer (RC). Therefore, the experiment consisted of 12 sets as follows: 3 t·ha−1 of the biofertilizer with 50, 75, and 100% of N and P; 4 t·ha−1 of the biofertilizer with 50, 75, and 100% of N and P; and 5 t·ha−1 of biofertilizer with 50, 75, and 100% of N and P, with 4 replications. The experiment was carried out in the greenhouse for 85 days.A two factorial experiment on sesame cultivation was designed in completely randomized blocks for adding the solid PNSB biofertilizer with four levels of 0, 3, 4, and 5 t·ha(1) Sesame Growth Parameters. The plant height (cm) was measured from the soil surface to the highest point of the plants. The number of leaves was counted as the total number of leaves on the main stem. The length of leaves was determined from the leaf collar to the leaf tip. The width of leaves was determined at the largest size of a leaf. The number of branches was counted from the bottom to the top, with the length of branch at least 3 cm. The number of capsules per plant was counted at harvest. The length of capsule was determined between two tips of a capsule. The dry biomass: Components of stem-leaf, shell, and seed were dried at 70°C for 72 h and the dry biomass was weighed. All parameters were measured according to Fazeli Kakhki et al. [i et al. .(2) Biochemical Parameters. The leaf chlorophyll content was determined by Moran [th leaf from the soil surface of 5 plants was collected after 30 days of sowing. A total of 1 cm2 of each leaf was added to each Eppendorf containing 3 mL·N,N-dimethylformamide for 24 h in an incubator at 25°C. The solution was determined by a spectrophotometer at the wavelengths of 664 and 647 nm for detecting chlorophyll a and b, respectively. Afterwards, chlorophyll a and b and total chlorophyll contents  were calculated as follows:by Moran . The 10tth leaf from the soil surface by SPAD-502 Plus according to the guide of the Konica Minolta manufacturer.The Soil Plant Analysis Development (SPAD) index was directly detected on the 12(3) Agronomic Traits. Sesame yield components and yields were determined on the capsule length, capsule diameter, number of capsules plant−1, number of filled capsules plant−1, percentage of capsules plant−1, number of seeds capsule−1, number of filled seeds capsule−1, percentage of filled seeds capsules−1, and a-thousand-seed oven-dry weight.total, Ptotal, and Ktotal Concentrations in Sesame at Harvest(1) Analysis of N. The stem-leaf, shell, and seed samples of each set were collected and dried at 70°C for 72 h. Then, they were ground to pass through a 0.5 mm sieve for the total analysis of all parameters. The Ntotal, Ptotal, and Ktotal in stem-leaf, shell, and seed were investigated. The total N, P, and K concentrations were determined by following the methods of Houba et al. [total was determined by the regular Kjeldahl method, the Ptotal concentration was determined by colorimetric procedure at the wavelength of 880 nm, and the Ktotal content was determined by using the ICP-OES.a et al. . The Ntototal, Ptotal, and Ktotal Accumulation in Sesame at Harvest(2) Determination of N. The amount of N, P, and K uptake was calculated from stem-leaf, shell, and seed biomass multiplied by each concentration of each component. The total uptake was calculated from all parts.P < 0.05.The data shown in this paper are mean values of the four replications. The data were subjected to the two-way analysis of variance (ANOVA) using the SPSS software package version 13.0. The significant differences were assessed by Duncan's post hoc tests at −1 of the solid PNSB biofertilizer was significantly higher than the control (63.4 cm). This led to a much higher number of leaves as well. However, no improvement in length and width of leaves by the solid PNSB biofertilizer was observed. For the chemical fertilizer, the reduction in amounts of N and P significantly decreased the plant height, number of leaves, and width of leaves, i.e., the plant height of the treatment applied with 50–75% N and P of the RC achieved 63.5–66.2 cm, and it was lower than the 100% N treatment, 68.6 cm  . The pla 68.6 cm .a and the total chlorophyll were higher in treatments applied with 75–100% N and P of the RC as compared with the one with 50% N and P of RC, with 10.7–10.9 and 8.78 µg·Chl·mL−1 and 13.7–13.9 and 11.2 µg·Chl·mL−1. The SPAD value also showed the same trend  in the treatments applied with the solid PNSB biofertilizer, compared with 0.14, 0.15, and 0.86% DW showed by the control (no added PNSB biofertilizer). The P concentrations showed a similar trend, with an exception for P content in seed. The K concentration was in the same pattern with the N concentration, its value with and without application found in stem-leaf (4.84–4.98 and 4.24% DW), shell (1.74–2.44 and 1.57% DW), and seed (0.15-0.16 and 0.12% DW). Increasing of N and P fertilizers input also enhanced N and P concentrations in seeds to 0.86, 0.90, and 0.96% N and 0.74, 0.76, and 0.76% P as in the order of 50, 75, and 100% N and P of the RC  produced a positive effect on the N concentration, with higher f the RC .−1), shell (6.62–6.95 g·pot−1), and seed (11.4–13.3 g·pot−1) compared with the no application (control) with 7.67, 5.75, and 9.4 g·pot−1, respectively . It is a similar trend to the potential acidity of pHnd pHKCl .−1 as 98.2∼87.8 < 128.2 < 136.5 mg NH4+ kg−1, whilst in the chemical fertilizer, results were recorded as 126.2 mg NH4+ kg−1 for 100% of the RC, 108.3 mg NH4+ kg−1 for 75% of the RC, and 103.7 mg NH4+ kg−1 for 50% of the RC , Fe-P , and Ca-P  concentrations, respectively  is proper for use as a biocompost in agriculture, while that of this study was 28.7.Increasing chemical fertilizers resulted in higher sesame growth and yield Tables  and 5. NR. palustris TLS06, VNW02, VNW64, and VNS89 strains has been reported for their impacts on rice growth and productivity in an application as a biofertilizer [R. palustris PSB06 strain stimulated the rice growth at the seedling stage by root elongation of seeds [R. palustris also increased the fresh biomass of rice seedlings [R. palustris TLS06, VNW02, VNW64, and VNS89 strains encouraged sesame growth by inoculating into seeds and also in a form of a solid mixed biofertilizer into the soil. Moreover, the R. palustris inoculation might help recovery of the soil community to reduce adverse effects caused by chemical fertilization [The PNSB promoted sesame growth by acting as biofertilizers and plant growth promoters that induced plants by releasing substances such as ALA and IAA into the soil . The rolrtilizer , 27, 33.of seeds , and theeedlings  and riceeedlings , 30. Thulization .2 from the air, so the treatments of the biofertilizer application obtained higher values than those without PNSB biofertilizer application. Similarly, the PNSB from the solid biofertilizer solubilized the insoluble P fractions in the AS in dykes. Thus, the biofertilizer application treatments produced more nutrients than those without PNSB biofertilizer treatments  from treatments of the solid biofertilizer was significantly higher than without PNSB biofertilizer treatment, 1.94 MPN·g−1 DSW. Besides, the application at 3–5 t·ha−1 of the solid PNSB biofertilizer supplied much organic matter from the SRS, resulting in higher contents in comparison with the control (R. palustris TLS06, VNW02, VNW64, and VNS89 strains can ameliorate soil quality by reducing the toxicities of Al3+ and Fe2+ concentrations by producing exopolymeric substances and siderophores [4+ and PO43− in acid sulfate soils [2+ toxicity [R. palustris PI5 strain and Rhodobacter sp. [Rhodobacter sphaeroides SC01 strain [The application of the solid PNSB biofertilizer mixture resulted in a remarkable improvement in the soil characteristics with higher concentrations of ammonium and soluble P by the activities of N fixation and P solubilization, respectively. It is obvious that the PNSB strains from the biofertilizer fixed Neatments . Accordi control . The TLSand anfG . These Pand anfG , 27, 33.and anfG , PNSB st control . This merophores , 23 and toxicity  and toxicter sp.  or the R1 strain .R. palustris TLS06, VNW02, VNW64, and VNS89 strains showed a promising application as a biofertilizer to improve soil properties, nutrient uptake, and crop yield. Thus, in the application at 4-5 t·ha−1 of the solid biofertilizer containing R. palustris, TLS06, VNW02, VNW64, and VNS89 strains plus 75% N and P of chemical fertilizers produced the seed yield of 13.7–13.9 g·pot−1, which was equal to that of the 100% N and P of chemical fertilizers. Hence, the solid PNSB biofertilizer contributed to reducing 25% of the chemical fertilizers.This PNSB group in the solid mixed biofertilizer produced more available plant nutrients in the soil, so sesame can absorb, contributing to improving the N, P, and K uptake. The percentage of N, P, and K accumulation significantly increased by 29.4–51.0, 22.4–37.3, and 30.8–50.7%, respectively Figures 1. To sum −1 of SRS containing the biofertilizer of Rhodopseudomonas palustris TLS06, VNW02, VNW64, and VNS89 strains in corporation with the chemical fertilizer significantly improved the growth of sesame based on agronomic parameters compared with the control with no PNSB biofertilizer. There was a remarkable increase in the N, P, and K uptake in sesame according to the application of the biofertilizer. The sesame seed yield increased along with the increase in the solid mixed PNSB biofertilizer levels, with 12.3 g·pot−1 at 3 t·ha−1, 14.5 at 4 t·ha−1, and 14.3 at 5 t·ha−1 compared with the control, 10.3 g·pot−1. Significant increases in the pH value, ammonium, soluble phosphorus, organic matter, and exchangeable potassium indicated that the fertility of the alluvial soil was improved by the solid mixed PNSB biofertilizer for the sustainable sesame cultivation in dykes.The application at 3, 4, and 5 t·ha


Although topical drugs are the mainstay of treatment for patients with mild‐to‐moderate psoriasis, the developments observed in this field in the last two decades have been limited. The most commonly used drugs are still vitamin D analogues and corticosteroids, both with several limitations. The aryl hydrocarbon receptor (AhR) plays a role in the pathogenesis of psoriasis, and tapinarof, a novel, first‐in‐class, small molecule topical therapeutic AhR‐modulating agent has been recently approved by the FDA for the topical treatment of plaque psoriasis in adults. Two large, 12‐week, phase III trials, PSOARING 1 and 2, showed that 35.4%–40.2% of patients in the tapinarof 1% cream arm achieved the primary endpoint  compared with 6.0%–6.3% for vehicle arm, respectively. The most common adverse effects were folliculitis, contact dermatitis, headache and pruritus. In the open label, 40‐week, extension trial, PSOARING 3, the efficacy and safety results were similar, with 40.9% of patients achieving a PGA = 0 at least one time during the trial and 58.2% of patients with PGA≥2 achieved PGA = 0/1 at least once during the trial, without tachyphylaxis. There were no new safety signals, with most frequent adverse events being folliculitis, contact dermatitis, and upper respiratory tract infection. Tapinarof 1% cream has shown to be effective and to have a favorable safety profile in the treatment of psoriatic patients, representing an alternative to the current therapeutic options, increasing our armamentarium in the topical treatment of psoriasis. A search in the PubMed database (up until June 2022) for articles with the specific keywords: “psoriasis,” “tapinarof,” “GSK2894512,” “aryl hydrocarbon receptor,” and “topical treatment” were carried out. The articles were selected by the relevance of the abstract and established objectives. When pertinent, the bibliographic references present in the selected articles were also included.2Aryl hydrocarbon receptor (AhR) is a cytosolic ligand‐dependent transcription factor expressed in different types of skin cells .AhR plays a role in the pathogenesis of inflammatory skin diseases.In psoriatic skin, the aryl hydrocarbon receptor is present in cutaneous plaques and regulates the inflammatory response as well as the terminal differentiation of keratinocytes and T lymphocytes.Tapinarof activates the nuclear factor erythroid 2‐related factor 2 (NRF‐2) pathway and consequently produces antioxidative enzymes that limit oxidative stress Figure .6, 10In conclusion, there is growing evidence that AhR plays a vital role, beyond the one present in skin homeostasis and integrity, as a potential therapeutic target in inflammatory diseases.3Heterorhabditis with insects. This nematode carries in its gut gram‐negative bacteria with luminescence properties called Photorhabdus luminescens, that is, released when the nematode infects an insect, killing it, by producing metabolites.P. luminescens produces metabolites with antibacterial properties that responsible for the observed biological action.Tapinarof is a topical aryl hydrocarbon receptor modulator of bacterial origin.Tapinarof cream 1% (GSK‐2894512) has the same active principle as benvitimod cream 1% (WBI‐1001). Benvitimod was developed in China, via a separate clinical trials program. It requires a twice daily application has a different excipients/formulation and is approved for topical use in China since 2019 for patients with mild‐to‐moderate plaque psoriasis.3.1Cmax) for tapinarof 2% and 1% was 2.5 and 1.2 ng/ml, respectively. At day 21 it was 0.3 ng/ml for the 2% cohort and 0.15 ng/ml for the 1% cohort. The value of the median time to reach the maximum serum concentration (Tmax) for both concentrations of tapinarof cream were between 1 and 4 h.A phase I study evaluated the pharmacokinetics (PK) of tapinarof cream 1% and 2% applied twice daily in 11 participants with atopic dermatitis. It was shown that a higher concentration of tapinarof cream leads to greater plasma concentrations of the drug, which tend to decrease over time which proves there is no accumulation after the repeated exposure. On day 1, the mean maximum serum concentration , a non‐invasive method limiting the use of skin biopsies. The cream was applied for 7 days, and images were taken every day before the drug administration. It was shown that in the first few days, the cream remained in the superficial layers of the skin, at day 10, there was no fluorescence detected.Tapinarof metabolism is hepatic, with cytochrome (CYP) P450 playing a predominant role. The primary enzymes involved are CYP1A2 and CYP3A4. Conversely, CYP2C9, CYP2C19, CYP2C20, CYP2D6 play minimal roles in the oxidative metabolism of tapinarof in hepatocytes.3.2a ≥ 1 grade PGA improvement, and six patients had a ≥ 2 grade improvement compared to day 1. Four patients (21.1%) achieved a PGA score of 0 or 1 and ≥2 grade improvement in PGA. Also, on day 29, there was a significant mean change in PASI score to 15.14 (95% CI – 19.61 to – 10.66). Eighteen patients improved their PASI score during this trial. Seven patients achieved PASI75. On day 1, the mean percentage BSA was 27.20 (SD: 7481). On day 29, there was a change in the mean percentage BSA to 14.44 (95% CI – 19.84 to – 9.04). Nineteen completed the trial. The reasons for discontinuation were “lost to follow‐up” in one patient and withdrawal from the study in another.A phase IIa, multicenter, open‐label trial NCT04042103)  Table  includedp < 0.001). PASI 75 was reached at week 12 in 65% (p = 0.001) and 56% (p < 0.001) in the tapinarof 1% group, twice or once daily, respectively. In the 0,5% tapinarof group PASI 75 was achieved in 46% in both twice or once daily applications . In the control group, 16% (twice daily) and 5% (once daily) reached PASI 75. For PASI 90 39% (p = 0.002) and 40% (p = 0.001) of participants in the 1% tapinarof group, and 31% (P = 0.008) and 18% (P = 0.057) of participants in the 0.5% tapinarof group, twice or once daily respectively. In the vehicle group, 0% of participants reached PASI 90 at week 12. In addition, changes in the mean PGA from baseline and the PGA response rate, was more significant in all tapinarof groups, and maintained for 4 weeks after the end of the study. Lastly, in evaluating the total target lesion grading scores from baseline, there was a higher reduction in the tapinarof groups than vehicle, beginning at week 2 and maintained until week 16.Additional outcomes were evaluated in a phase IIb Table  study, wp < 0.001 vs. vehicle) and 6.0% for vehicle cream. In PSOARING 2, the percentage of subjects achieving the primary endpoint was 40.2% for tapinarof 1% cream (p < 0.001 vs vehicle) and 6.3% for vehicle cream. Overall, in the two studies, at week 12, PASI 75, a PGA score of 0 or 1, BSA changes and PASI 90 were observed in a significantly higher proportion of patients in the tapinarof 1% cream arm.Two phase III multicentre, randomized, double blind, vehicle‐controlled trials, PSOARING 1 (NCT03956355) and PSOARING 2 (NCT03983980), included participants aged between 18 and 75 years old with mild‐to‐severe plaque psoriasis with stable disease for at least 6 months and BSA between 3% to 20% and a PGA score of at least of 2. PSOARING 1 (Table n = 312), the total duration of remittive effect off therapy was a mean of 130.1 days.Recently, the first long‐term results were published from the PSOARING 3 trial. This open label, extension trial with 763 patients aimed to explore the one‐year efficacy, safety, tolerability, durability of response on therapy (absence of tachyphylaxis), and duration of remittive effect off‐therapy of tapinarof cream 1% in plaque psoriasis. Patients received a maximum of 40 weeks of open‐label treatment, followed by 4‐weeks' off‐treatment follow‐up after being enrolled in PSOARING 1 and 2 in the tapinarof or vehicle arm. Patients could receive tapinarof for up to 52 weeks from PSOARING 1 and 2 start  through PSOARING 3 conclusion (40 weeks). In PSOARING 3, patients were treated according to their PGA score. Patients presenting at the beginning of PSOARING 3 with complete disease clearance (PGA = 0 [clear]), discontinued treatment and were monitored for remittive effect . Patients entering with PGA≥1 maintained application of tapinarof to all affected areas, including newly appearing lesions. All patients that achieved PGA = 0 stopped treatment and were observed for remittive effect off therapy. If a disease flare occurred, defined as PGA ≥ 2, then treatment was re‐started and continued until PGA = 0. At baseline, 10.4% of patients had PGA = 0 [only 2.0% (5/255) were patients previously treated with vehicle]. Overall, 40.9% achieved complete disease clearance (PGA = 0) at least one time during the trial. Also, 58.2% of patients that entered PSOARING 3 with PGA≥2 achieved PGA = 0 or 1 at least once during the trial. No tachyphylaxis was demonstrated for up to 52 weeks. Among patients achieving PGA = 0 at any time during the trial   Table , the treIn a phase IIb trial, the adverse effects reported had mild‐to‐moderate severity. It was applied tolerability scores considering the participants and the investigator's point of view for 12 weeks. Participants scored 0 to 1, which means none or slightly site burning and itching on the application site. The investigators scored mostly 0.In the PSOARING 1 and PSOARING 2 phase III trials  showed that after 52 weeks of treatment there were no new safety signals. The most frequent treatment‐emergent adverse events included folliculitis (22.7%), contact dermatitis (5.5%), and upper respiratory tract infection (4.7%). The incidence and severity of folliculitis and contact dermatitis was not worse with long‐term treatment compared with the pivotal trials, and most of these adverse events were mild or moderate.4Psoriasis is a common disease that disrupts daily activities and consequently has a significant impact on quality of life.The aryl hydrocarbon receptor is a cytosolic ligand expressed in several skin cell types. AhR signaling is required in healthy skin to maintain skin homeostasis by regulating the skin immunological network, keratinocyte differentiation, skin barrier function, and gene expression in cells. In psoriatic skin, there is a higher expression of AhR.Tapinarof, an AhR agonist, is a recently FDA approved non‐steroidal topical treatment for plaque psoriasis. Its mechanism of action includes suppression of inflammatory cytokines (in particular IL‐17), the modulation of skin barrier protein expression, and antioxidant activity.Tapinarof showed to be effective in phase II and phase III randomized controlled trials in patients with mild‐to‐moderate plaque psoriasis. In the phase III trials PSOARING 1 and 2 a PGA response occurred in 35%–40% of patients in the tapinarof group; while only in about 6% of patients in the vehicle group.Most adverse events associated with tapinarof were cutaneous, including pruritus, folliculitis, and contact dermatitis. Headache was a frequently reported as adverse effect. Nevertheless, the intensity of these adverse events was mild to moderate with spontaneous resolution and rarely led to the discontinuation of the treatment. No systemic drug accumulation has been reported. There was no increased risk of severe adverse effects, such as cardiac issues or prolongation of QT interval, up to the 52‐week period.Finally, it is important to highlight that treatment adherence to topical therapies is recognized as a critical issue in the management of chronic diseases such as psoriasis, particularly influencing treatment efficacy.In summary, tapinarof 1% cream has been shown to be effective and safe in the treatment of psoriatic patients, representing an alternative to the current therapeutic options, increasing our armamentarium in the topical treatment of psoriasis.Sofia Nogueira, Maria Alexandra Rodrigues, Ron Vender and Tiago Torres had the idea for the article, performed the literature search and data analysis, and drafted and critically revised the work.Sofia Jacques de Nogueira and Maria Alexandra Rodrigues declare no potential conflict of interest. Ron Vender has been a consultant, and/or scientific advisor, and/or investigator, and/or speaker for Amgen, AbbVie, Arcutis, Astellas, Bausch Health/Valeant, BMS, Boehringer Ingelheim, Celgene, Dermira, Eli Lilly, Galderma, GSK, Janssen, Leo Pharma, Merck (MSD), Merck Serono, Novartis, Pfizer, Regeneron, Roche, Sanofi‐Aventis/Genzyme, Sun Pharma, Takeda and UCB. Tiago Torres declares research grants and/or consulting fees from AbbVie, Almirall, Amgen, Arena Pharmaceuticals, Biocad, Boehringer Ingelheim, Bristol‐Myers Squibb, Celgene, Eli Lilly, Janssen, LEO Pharma, MSD, Novartis, Pfizer, Samsung‐Bioepis, Sandoz, Sanofi, and Viatris.


Oudemansiella raphanipies polysaccharides (ORPs). The results showed that the optimal extraction parameters were an alkali concentration of 0.02 mol/L, a ratio of material to liquid of 1:112.7 g/mL, an extraction temperature of 66.0 °C, and an extraction time of 4.0 h. Under the optimal conditions, the yield of ORPs was raised to 16.2 ± 0.1%. The antioxidant activities of ORPs–I~V were determined and compared, and ORPs–V was further purified by chromatography, with an average molecular weight (Mw) of 18.86 kDa. The structure of ORPs–V was determined by Fourier transform–infrared spectroscopy (FT–IR), monosaccharide analysis, and nuclear magnetic resonance (NMR) spectroscopy. The ORPs–V comprised fucose, rhamnose, arabinose, glucose, galactose, mannose, xylose, fructose, galacturonic acid, and glucuronic acid at a ratio of 1.73:1.20:1.13:2.87:8.71:2.89:1.42:0.81. Compared to other ORPs, ORPs–V showed the strongest antioxidant activities , and were able to significantly increase the activities of superoxide dismutase, catalase, lactate dehydrogenase, and glutathione peroxidase. However, they reduced the malondialdehyde content in mice fed a high-fat diet. These results indicate that ORPs–V may be good anti–oxidant agents to be applied in functional foods.Response surface methodology (RSM) was used to find the optimal extraction process of  Oudemansiella raphanipies (O. raphanipies) has been described uniformly as either O. radicata or O. furfuracea, and, until recently, was identified as O. raphanipies on the basis of findings from morphological characteristics and molecular phylogenetic studies  (1% w/v in deionized water), respectively. The mixed solutions were maintained at 37 °C for 25 min, and the above solutions were added into 500 μL of trichloroacetic acid (10% w/v in water) and centrifugated at 3000× g  for 15 min at 25 °C. The supernatants (500 μL) were blended with 500 μL of deionized water and 0.5 mL of FeCl3 (0.1% w/v in water), and then the absorbance was determined at 700 nm against the blank control group, which consisted of all reagents except the ORPs.sample is the absorbance of the polysaccharides and Ablank control is the absorbance of the negative control group.The reducing power of ORPs was analyzed according to the method reported by Chen et al. . In brieThe anti–oxidant activities of ORPs–V in vivo were evaluated according to the method of Zeng et al. . BrieflyT-test (p = 0.05) or a Fischer’s F-test (p = 0.01 or 0.05). All operations and experiments were conducted 3 times, and all of the data are shown herein as mean ± SD. The statistical analysis results of the single–factor data were obtained by Origin 9.0  and SPSS13.0 software . The significance of differences among different experimental groups was evaluated based on a one–way analysis of variance (ANOVA). Finally, significant differences were evaluated using an independent sample As a traditional extraction method for polysaccharides from natural products, the hot extraction method offers many advantages, including low cost, convenient operation, and no special equipment required, over other separation methods. Thus, it is suitably used for large–scale industrial applications .p < 0.05) with the increase in alkali concentration (from 0.01 to 0.02 mol/L), and reached its maximum (15.8% ± 0.9%) when the alkali concentration was 0.02 mol/L. However, as the alkali concentrations further increased (from 0.03 to 0.06 mol/L), the extraction rate significantly decreased (p < 0.05) and then gradually became constant, as shown in Tremella fuciformis polysaccharides prepared by alkali water extraction (10.1~20.2%) [It has been confirmed that natural polysaccharides could be extracted with alkaline solutions to maximize material utilization and expand the extraction yield. To determine the effects of various alkali concentrations on the extraction rates of ORPs, the extraction was conducted at alkali concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mol/L, and other conditions were kept as below: a ratio of material to liquid of 1: 100.0 g/mL, an extraction temperature of 70.0 °C, and an extraction time of 4.0 h. The results showed that the extraction rate of ORPs dramatically increased (1~20.2%) ,32.The influence of the ratios of different materials to liquid on the extraction rate of ORPs is shown in To investigate the influence of temperature on the extraction rate of ORPs, the extraction temperature was set to 50.0, 60.0, 70.0, 80.0, and 90.0 °C, respectively. All other conditions were set as follows: alkali concentration of 0.01 mol/L, ratio of material to liquid of 1: 100.0 g/mL, and extraction time of 4.0 h. Finally, the effects of various extraction times on the extraction rate of ORPs is indicated in To summarize, the alkali extraction method can be applied to enhance the extraction efficiency and reduce the separation time of acidic ORPs, but the amount of alkali added should be paid attention to and strictly controlled. In addition, common influencing factors should not be overlooked either, including the material–to–liquid ratio, temperature, and time.X) and the independent response variable (Y): X1 is the alkali concentration (mol/L), X2 is the ratio of material to liquid (g/mL), X3 is the extraction temperature (°C), and X4 is the extraction time (h). The test data were processed according to the multiple regression analysis, as given below. A second–order polynomial modeling method worked out the regression analysis on the basis of the relationship between the dependent response variable , which suggested that the model fit well. Thus, the reliability of the model was verified. Furthermore, the values of coefficient of determination (R2) and the adjusted determination coefficient (R2adj) indicated that the model fit well with the experimental data and the theoretical values of the extraction rate of ORPs, which were 0.9963 and 0.9926, respectively. Furthermore, the lack of fit (p > 0.05) suggested that there a failure occurred with a model representing the test data. The chances of a “lack of fit–value” due to noise were remote. As a result, the results indicated a good fit. An extremely low coefficient of variation value (C.V. = 16.16%) represented the high reproducibility and reliability of the experimental values. As shown in X1, X2X3, and X4); quadratic term coefficients ; and cross–product coefficients  were all significant (p < 0.05). The F value showed that the order of factors influencing the ORP extraction rate was ratio of material to liquid > extraction temperature > extraction time > alkali concentration, and the order of the interaction effects was X2X3 > X1X2 > X2X4 > X1X3 >X3X4 > X1X4.Based on the ANOVA results for this model indicated in In order to directly observe the influence of independent variables on the response, three–dimensional (3D) response surface plots were obtained for the purpose of quantifying the optimal values in order to achieve the optimal extraction rate of ORPs. Both the 3D response surface plots  and contThe influences of alkali concentration and the ratio of material to liquid; alkali concentration and extraction temperature; and alkali concentration and extraction time on the extraction rate are shown in The effects of the ratio of material to liquid and extraction temperature and the ratio of material to liquid and extraction time on the extraction rate are illustrated in As indicated in p > 0.05). All of the results indicate that the experimental model used in this study was effective.The optimal conditions for maximizing the extraction rate of ORPs were obtained from the model: alkali concentration, 0.02 mol/L; ratio of material to liquid, 1:112.7 g/mL; extraction temperature, 66.0 °C; and extraction time, 4.0 h. The predicted point was experimentally verified under the actual optimum conditions: alkali concentration of 0.02 mol/L, ratio of material to liquid of 1:113.0 g/mL, temperature of 66.0 °C, and time of 4.0 h. Under the optimum conditions, the extraction rate of ORPs was 16.2 ± 0.1%, and no significant differences were found with which to compare the predicted value of 16.3% , dialyzed, deproteinated, and freeze–dried to prepare the crude ORPs, and ORPs–I~ORPs–V was obtained by precipitation with different concentrations of ethanol , respectively. Because the crude polysaccharides may contain various impurities, including protein, monosaccharide, and other small molecule compounds, ORPs–V was further purified by the ion–exchange column DEAE–52 and Sephadex G–150 columns. As shown in An extract, in an alkaline solution of polysaccharides from Mw significantly affects the biological activities of polysaccharides. HPGPC was used to determine the Mw of ORPs–V. According to the equation of standard curve, Log Mw = −3.5369 T + 33.437 (R2 = 0.9994), . The Mw of ORPs–V was calculated to be 18.86 kDa, and the retention time was 20.35 min. As shown in the HPGPC spectrum, a single and symmetrical peak for ORPs–V was obtained, indicating that the purified ORPs–V was homogeneous polysaccharide . The 13C–NMR spectrum of ORPs–V had a clearer chemical shift and better resolution than 1H–NMR. In addition, the wide signal at 3.50~4.00 ppm was attributed to galactose residues, and the signal peaks between 3.35 and 4.40 ppm were regarded as the protons of the CH2–O and CH–O groups on the sugar rings. The signal peaks at 4.53, 4.93, and 5.02 ppm were due to mannitol and glucose [According to the results of the FT–IR analysis, there were no apparent differences in the ORPs. Thus, only the ORPs–V fraction was further investigated using charides ,43,44. Tguration . In the analysis . The sig2 to C6, and δ 90.00~110.00 ppm was the signal for the anomeric carbon area. ORPs–V contained three major types of anomeric carbon signals. The anomeric carbon signals at δ 100.08, 102.16, and 103.48 ppm were observed in the 13C–NMR spectrum. The lower–field chemical signals, in the range of 175.89 ppm, showed that there were some uronic acids in the ORPs. The overlapped signals of C2~C6 were seen in the range of δ 60.00~80.00 ppm. No absorbance at δ 80.00~90.00 ppm was observed in the 13C–NMR spectrum, which indicated that there was no furanose in ORPs–V. The chemical shifts of anomeric protons below 4.77 ppm and carbon resonance above 100.45 ppm indicated that the Galp residues were β–linked–anomeric [The δ 60.00~85.00 ppm value was attributed to the chemical signal peaks of C1H–NMR and 13C–NMR spectra [13C/1H–NMR was applied to further evaluate the structural properties of the ORPs–V. As indicated in 13C/1H cross peaks at 102.71/4.37, 71.07/3.43, 78.23/3.64, 71.54/3.51, and 69.19/3.93 ppm, which were seen as the C1–H1, C2–H2, C3–H3, C4–H4, and C5–H5 of the β–D–galactose residues, respectively. Some cross–peaks could be attributed to Xylp units, which were also observed in the HSQC spectrum. For instance, the spectra indicated the presence of HSQC 13C/1H cross peaks at δ 73.70/3.42 and 61.60/3.78 ppm, which may be attributed to C3 and C5 of the β–1, 3, and 4–linked Xylp residues. The structural elements of the β–T–linked Xylp units were observed in the spectra of HSQC at δ 99.10/4.48 (C1–H1), 72.00/3.31 (C2–H2), and 69.20/3.62 (C4–H4) [Identifying the chemical linkages in the natural polysaccharides was highly difficult, because the signal peaks of the fractions overlapped severely in the  spectra . As a re (C4–H4) . Neverth·+, hydroxyl, DPPH radial, and reducing power tests, which are displayed in Free radicals pose a major risk to the health of the human body and play a vital role in different kinds of lifestyle–related diseases, including aging, heart disease, cancer, arteriosclerosis, and diabetes. Antio–xidants are crucial because they can eliminate free radicals from the body, which may harm DNA and proteins. Polysaccharides are generally regarded as the main active ingredients in mushrooms. The anti–oxidant activities of the ORPs–I~ORPs–V were evaluated in vitro by ABTS+–scavenging assays have been generally applied to test the anti–oxidant capacity of natural polysaccharides. ABTS+ could react with K2(SO4)2 to form stable glaucous cation radicals, which would be measured at 734 nm [·+ were enhanced in a dose–response pattern, which indicated the presence of significant scavenging activity at all concentrations. Compared to other ORPs, ORPs-V showed better scavenging activity, second only to Vc. The EC50 values of ORPs–I, ORPs–IV, and ORPs–V were far lower than 0.5 mg/mL, respectively. However, both ORPs-II and ORPs-III showed relatively weaker scavenging activity, and their EC50 was approximately 0.5 mg/mL. The hydroxyl radical–scavenging activities of the ORPs are shown in 50 values of ORPs–IV and ORPs–V were significantly lower than 0.5 mg/mL. Both ORPs–II and ORPs–III showed weaker hydroxyl radical–scavenging activities, because the EC50 values of both were greater than 1.0 mg/mL. It was noteworthy that ORPs–I~ORPs–V showed significant scavenging activities for DPPH, but their EC50 value was much lower than 0.5 mg/mL , indicating that the HFD model mice had been under severe oxidative stress caused by their high–fat diets. Interestingly, after the treatment with ORPs–V at a dose of 200 mg/kg/d, the enzyme activities of SOD, CAT, GSH–Px, and LDH in the ORPs–V–H group were significantly increased to 29.5 ± 0.4, 7.9 ± 0.3, 11.1 ± 0.3, and 68.7 ± 0.9 U/mL, respectively, when compared with those in the HFD model control groups (p < 0.01). Most notably, the enzyme activities of GSH–Px in the ORPs–V–H group were significantly increased, markedly higher than the NC group (p < 0.01). In addition, the contents of MDA in the ORPs–L, ORPs–M, and ORPs–V groups were markedly reduced when compared with those in the HFD model control groups (p < 0.01). Notably, no significant differences were found between the ORPs–V–H group and the NC group (p > 0.05). The above results indicate that ORPs–V can effectively alleviate HFD–induced oxidative stress, and a high dose of ORPs–V performs better activities than a low dose. More in–depth studies should be conducted to elucidate the detailed mechanism behind this effect.Oxidative stress has been widely considered as a central player in various chronic metabolic diseases, including obesity, diabetes, cancer, cardiovascular disorders, and inflammatory diseases . SOD, CAMw were better than those of other fractions, and the anti–oxidant activities of ORPs–V in vivo were also confirmed. Interestingly, ORPs–V with higher contents of glucose, galactose, and mannose seemed to have better anti–oxidant activities. Previous studies have shown that Mw in polysaccharides may be an important factor in anti–oxidant activities. Moreover, monosaccharide composition, type of glycosidic linkage, the amount of hydroxyl group, or chain conformation of polysaccharides seem to be closely correlated to polysaccharides’ anti–oxidant activities. For instance, the polysaccharides with galactose, glucose, and glucuronic acid showed stronger anti–oxidant activities than those without them [Mw, the branch, the microstructure, and the anti–oxidant activities of ORPs remains to be carried out.To summarize, these results indicate that the ORPs–I~ORPs–V have different degrees of anti–oxidant activities in a concentration–dependent manner, and the experimental results of four assays in vitro were similar. The anti–oxidant activities of ORPs–V with lower out them . The polout them . In addiout them . The monβ–Gal, with branches consisting of β–Glcp–(1→6), β–Manp–(1→6), β–Manp–(1→2), Araf–, and β–Xylp–(1→). However, their molar ratio was significantly different, and the high content of glucose, galactose, and mannose seems to have had a positive effect on their anti–oxidant activities. Further studies should be undertaken to elucidate the structure–function relationship and molecular mechanism of ORPs and ORPs–V. These results indicate that ORPs–V may be used as a source of natural anti–oxidants in the food or pharmaceutical industries, and can provide an important reference for the comprehensive utilization of O. raphanipies. As a precious edible mushroom with a high nutritional value, which is mainly distributed in Hainan, Fujian, Jiangsu, and Yunan in China, ORPs are attracting great interest from researchers. In this study, the extraction rate of the ORPs was highly improved to 16.2% under the optimal conditions . All responses were statistically studied and found to fit with the quadratic regression model. The ratio of material to liquid was found to have the main effect on the extraction rate of ORPs, which was more important than the extraction temperature, extraction time, and alkali concentration. The anti–oxidant activities of ORPs, both in vitro and in vivo, were evaluated and classified using water extraction and ethanol precipitation methods. ORPs–V displayed the strongest antioxidant activity in vitro, and the animal experiments also confirmed the anti–oxidant activities of ORPs–V. In addition, the FT–IR, monosaccharide analysis, and NMR showed that ORPs–V consisted of L–fucose, L–rhamnose, L–arabinose, D–glucose, D–galactose, D–mannose, D–xylose, D–fructose, D–galacturonic acid, and D–glucuronic acid. The main backbone of the proposed AAP–V structure was composed of 1→3, 6 and 1→4, 6–


Staphylococcus aureus (S. aureus). Herein, a novel method, based on immunomagnetic separation using IMBs and recombinase polymerase amplification (RPA), was employed to detect S. aureus strains in milk and pork. IMBs were formed by the carbon diimide method using rabbit anti-S. aureus polyclonal antibodies and superparamagnetic carboxyl-Fe3O4 MBs. The average capture efficiency for 2.5 to 2.5 × 105 (CFU)/mL gradient dilution of S. aureus with 6 mg of IMBs within 60 min were a range of 62.74 to 92.75%. The detection sensitivity of the IMBs-RPA method in artificially contaminated samples was 2.5 × 101 CFU/mL. The entire detection process was completed within 2.5 h, including bacteria capture, DNA extraction, amplification, and electrophoresis. Among 20 actual samples, one case of raw milk sample and two cases of pork samples were tested positive using the established IMBs-RPA method, which were verified by the standard S. aureus inspection procedure. Therefore, the novel method shows potential for food safety supervision owing to its short detection time, higher sensitivity, and high specificity.Separation processes using immunomagnetic beads (IMBs) are advantageous for the rapid detection of IMPORTANCE Our study established IMBs-RPA method, which simplified the steps of bacteria separation, shortened the detection time, and realized the convenient detection of S. aureus in milk and pork samples. IMBs-RPA method was also suitable for the detection of other pathogens, providing a new method for food safety monitoring and a favorable basis for rapid and early diagnosis of diseases. Staphylococcus aureus is a Gram-positive opportunistic zoonotic pathogen that is commonly found airborne in outdoor environments, in the nasal cavity and skin of humans, and in some cases in food. S. aureus poses threats to human and animal health because it can cause a variety of diseases, such as purulent infection (S. aureus (MRSA) has also been detected in milk, dairy products, and meat in recent years  suitable for biomedical, data storage, spintronics, catalytic, neural stimulations, and gyroscopic sensing applications   , real-tion (PCR)  and Multon (PCR)  are common (PCR)  and enzyon (PCR)  are alsoRecombinase polymerase amplification (RPA) is a new DNA amplification technology proposed by Piepenburg et al. in 2006. Within a short span of time since then, RPA has been widely used in various applications, such as in the diagnosis of human and animal infectious diseases –35, dete–S. aureus from milk and pork samples .In this study, a novel, rapid, highly sensitive, and specific alternative method, based on the combination of immunomagnetic separation and RPA, was developed for the on-site detection of food pathogens. Immunomagnetic beads (IMBs) were prepared and then used to rapidly separate and enrich  samples . The res3O4 MBs. The Fe3O4 MBs were composed of highly aggregated smaller grains. On the other hand, the TEM image shown in 3O4 MBs as a function of the applied magnetic field measured using a vibration sample magnetometer. The saturation magnetization and coercivity of the Fe3O4 MBs were 74.37 emu/g and 40 Oe, respectively. Therefore, the synthesized Fe3O4 MBs exhibited a strong magnetic response and superparamagnetism.3O4 MBs. The characteristic absorption peak at 583 cm−1 can be attributed to the Fe-O bond. Peaks corresponding to the asymmetric (ν[COO-] asym) and symmetric (ν[COO-] sym) stretching vibrations of the carbonyl group were also observed at 1,536 and 1,411 cm−1, respectively. In addition, the characteristic absorption bands observed at 3,436, 2961, and 1,042 cm−1 can be attributed to the stretching vibrations of the O-H, C-H, and C-O bonds, respectively. Lastly, the absorption band at 1627 cm−1 corresponds to the out-of-plane bending vibration of the –OH group (δ OH···O out-of-plane).S. aureus polyclonal antiserum was 1:51,200 , and the maximum safe limit value is 1000 CFU/g (mL). Therefore, the sensitivity of the IMBs-RPA method established in our study can well meet the detection limit requirements in the national standard. After the isolation and enrichment of the S. aureus strains in artificially contaminated samples using IMBs, the detection line of PCR and RPA methods were both 2.5 × 101 CFU/mL. The IMBs specifically captured S. aureus in samples and separated them under the action of an external magnetic field, which realized the enrichment of S. aureus, and increased the template amounts that used for amplification, thus reduced the detection line and increased the sensitivity. So relative to the sensitivity of PCR method alone, a 100-fold increase was observed for IMBs-PCR and IMBs-RPA. Due to the high sensitivity of the RPA method, it is necessary to pay attention to the false-positive situation caused by aerosol contamination and to make a good partition between the reagent preparation, template addition, nucleic acid amplification, and product analysis areas during the RPA.The amplification efficiency of the RPA mainly depends on the selection of the target sequence and design of primers . The nucs strain . Eight pperature , 47. Her1 CFU/mL . On the nuc gene of S. aureus was developed. The proposed method simplified the pretreatment procedures for pathogen detection in food samples. As such, short detection time, higher sensitivity, and high specificity were achieved, which proves the potential of the novel route for food safety supervision.In this study, a novel and rapid detection method based on the combination of immunomagnetic separation and RPA for targeting the 3·6H2O), sodium acetate (NaAc), ethylene glycol, polyethylene glycol , and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. . 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-Morpholinoethanesulfonic acid monohydrate (MES), bovine serum albumin (BSA) and Gram Stain kit were purchased from Solaibao Biological Technology Co., Ltd. . Sodium azide (NaN3) was purchased from Sigma-Aldrich . Seven point five percent Sodium Chloride Broth, Blood agar plate, Brain Heart Infusion Broth and Freeze-dried rabbit plasma were purchase from Qingdao Haibo Biology Company, China.Ferric chloride hexahydrate (FeClS. aureus (ATCC29740), Salmonella Typhimurium , Escherichia coli , and Listeria monocytogenes  were preserved in the laboratory. L. monocytogenes strains were cultured in tryptic soy broth  at 37°C for 18 h. The other strains were cultured in Luria-Bertani broth (LB) at 37°C unless otherwise specified. The S. aureus strains captured by the IMBs was quantified at 37°C for 12 h using a Baird-Parker Agar , and those of the other strains were determined using the LB agar.The actual samples used in this study were collected in various districts of Beijing. Twenty raw milk samples were obtained from 10 different dairy farms, and chilled pork samples were obtained from 10 different farmers' markets in spring and summer.3O4 magnetic beads were synthesized by solvothermal method. First, 3.51 g of FeCl3·6H2O and 7.46 g of NaAc were separately added to 40 mL of ethylene glycol under vigorous stirring for 30 min. Then, mixed the two together and added ethylene glycol to make final volume to 100 mL, the resulting precursor solution was stirred vigorously for another 3 h, and then transferred into a high-temperature reactor to react at 200°C for 5 h, followed by cooling to room temperature. Finally, the black target product was dried in vacuo at 60°C for 8 h after washing three times with ethanol and water alternately.Monodisperse Fe3O4 MBs were measured at 25°C using a vibration sample magnetometer . Lastly, to characterize the chemical structures of the samples, Fourier-transform infrared (FTIR) spectroscopy  was performed over the wave number range of4000 to 400 cm−1.The morphology and structure of the samples were observed by field emission scanning electron microscopy  and field-emission transmission electron microscopy . The magnetic properties of the synthesized carboxyl-Fe9, 1 × 1010, 1 × 1010, 2 × 1010, and 2 × 1010 CFU (CFU) of heat-inactivated S. aureus on days 1, 4, 7, 14, and 21 of the experiment, respectively. Blood samples were collected from the heart 5 days after the last immunization. The anti-S. aureus polyclonal antisera were purified using a protein A antibody purification kit . The concentration, purity, and titers of the purified antibodies were determined by bicinchoninic (BCA) protein assay , sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and ELISA.Two female New Zealand White rabbits (2.25 kg) were immunized with 5 × 103O4 MBs were washed three times using MEST buffer  and then added 200 μL 0.1 M MES solution (pH 5.0) containing EDC (100 mg/mL) and NHS (100 mg/mL) successively. The MBs were activated through the rotary mixing of the resulting solution at 37°C for 1 h. Then, the components of the mixture were magnetically separated; the supernatant was discarded. The purified immunoglobulin (IgG) (0.5 mg) was added to the tube. The volume was made up to 500 μL through the addition of the MES. To obtain the IMBs, the resulting IgG-MB mixture was rotated for 1h at 37°C. The IMBs were blocked through incubation at 37°C for 1 h using 1 mL of 1% BSA (wt/vol) in 0.1 M MES buffer solution. Thereafter, they were washed thrice with 0.01 M phosphate-buffered saline (PBS) (pH 7.4). Finally, the IMBs (10 mg/mL) were resuspended in PBS containing 0.1% BSA (wt/vol) and 0.02% NaN3 (wt/vol) and then stored at 4°C.The carboxyl-FeS. aureus was diluted serially to prepare samples with cellular concentrations ranging from 2.5 × 10° to 2.5 × 105 CFU/mL in PBS. The IMBs were added to the serially diluted bacterial samples to a final concentration of 5 mg/mL and the blank control without the IMBs was set, followed by incubating the sample at 37 °C for 1 h with gentle rotation. The IMBs with the bound target bacteria were separated from the solution using a magnet, washed thrice with PBS to remove any unbound S. aureus strains, and then plated on the Baird–Parker agar medium to determine the capture efficiency of the IMBs on the target bacteria. Freshly cultured S. Typhimurium, E. coli, L. monocytogenes, and S. aureus were diluted to 104 CFU/mL with PBS. To investigate the specificity of the IMBs toward S. aureus, the capture and plating processes were performed as described above. All the agar plates were incubated at 37°C for 12 to 16 h. Capture efficiency was calculated through the formula, 0 and C1 represent the colony counts on the blank control and those bound to the IMBs, respectively.Freshly cultured nuc gene was used to design RPA primers, which encodes a thermostable nuclease unique for S. aureus strain (nuc gene of S. aureus was downloaded from GenBank. Eight primer pairs (Table S1) for RPA were designed according to the recommendations of the TwisDx Assay Design Manual , and evaluated to estimate which one can provide the best amplification. RPA reactions were carried out with the Twist@basic kit . RPA reaction system contains three enzymes, recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA-binding proteins (SSB), and strand-displaced DNA polymerases, these enzymes can affect the progress of DNA in polyacrylamide gel electrophoresis, lead to stripe dispersion. So the amplification products of RPA need to be purified by phenol-chloroform extraction and then visually detected via DNA gel electrophoresis. The RPA sensitivity was determined using DNA extraction of 2.5 × 104, 2.5 × 103, 2.5 × 102, 2.5 × 101, and 2.5 × 10°CFU/mL S. aureus cells. Meanwhile, the DNA of S. Typhimurium, E. coli, L. monocytogenes, and S. aureus were used to study the specificity of the RPA method. The genomic DNA was extracted using a bacterial genomic extraction kit  and detected using the RPA.The s strain . The seqS. aureus according to standard culture methods. S. aureus strain cultures with a final concentration of 2.5 × 104, 2.5 × 103, 2.5 × 102, 2.5 × 101, 10, 5, 2, and 1 CFU/mL were added to 10 mL of milk and pork homogenates. The S. aureus strains in such artificially contaminated samples were captured through immunomagnetic separation. Then, the genomic DNA of the S. aureus culture was extracted and detected using the RPA. To verify the results, PCR was performed with Taq DNA polymerase, and the reaction procedure was initial denaturation at 95°C for 5 min followed by 30 cycles denaturation at 95°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 30 s. The nuc-1.2 oligonucleotides (Table S1) were used as specific primers for PCR.Pasteurized milk and fresh pork samples purchased from supermarkets were found to be negative for S. aureus inspection procedure. Briefly, initial identification was carried out by the appearance of black single colonies on Baird-Parker plates and the transparent hemolytic rings on plates, and suspected colonies were further subjected to Gram staining and coagulase test . Before the sample analysis, milk and pork samples were diluted or homogenized using PBS in a ratio of 1: 9. The amplification results were analyzed through DNA gel electrophoresis.The established IMBs-RPA method was used to detect 20 raw milk and chilled pork each from different dairy farms and farmers' market. The results of IMBs-RPA were also verified by PCR and standard t test and two-way analysis of variance (ANOVA). Differences were considered statistically significant if P < 0.05.The experiments were performed in triplicates, and all measurements were conducted three times per experiment. Statistical analyses were performed using GraphPad software . Statistical significance was determined through the Student's All data generated or analyzed during this study are included in this published article .


Cyberlindnera fabianii (Cy. fabianii) planktonic cells and biofilms regarding the most frequently administered systemic antifungals, despite the high mortality rate and its potential role in catheter-related infections. In the current study, the activity of fluconazole, amphotericin B and echinocandins  was determined against planktonic and sessile cells of Cy. fabianii clinical isolates (n = 8). Planktonic minimum inhibitory concentrations (MICs) ranged from 1 to 2, from 0.25 to 1, from 0.015 to 0.06, from 0.03 to 0.12 and from 0.25 to 0.5 mg/l for fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin, respectively. One-day-old biofilms were highly resistant to fluconazole (MIC ranged from 512 to > 512) compared to planktonic counterparts, but not to amphotericin B (MIC ranged from 0.25 to 2 mg/l) and echinocandins (MIC ranged from 0.06 to 2 mg/l). Based on the calculated planktonic killing rates, the highest activity was observed in the case of anidulafungin , while micafungin, caspofungin, amphotericin B and fluconazole exerted 0.46–1.47, 0.14–0.86, −0.03 to 2.08 and −0.15 to 0.09 killing rate value ranges, respectively. The obtained in vitro planktonic and sessile susceptibility patterns suggest that echinocandins and amphotericin B may be the most reliable treatment option for the treatment of Cy. fabianii infections.Until recently, little was known about the susceptibility pattern of  Cy. fabianii is a rarely isolated pathogenic yeast, which is associated with endocarditis, prostatitis and fungaemia as well as catheter-related infections. In the past decade, the emergence of Cy. fabianii is steadily growing, presumably due to the increasingly usage of indwelling devices  assay, as described previously [C. krusei ATCC 6258 and C. parapsilosis ATCC 22,019 were within the accepted quality control (QC) ranges for all tested antifungals [Cy. fabianii; nevertheless, according to the epidemiological cut-off values (ECVs) published by Lee et al. [The MIC values for ifungals . There ae et al. , all tesk was 1.25) compared to the effect observed at concentrations ranging from 0.25–1 mg/l  , while the average k value was slightly higher in the case of micafungin at 0.25 mg/l (0.96 average 1/k) compared to the k values obtained at the higher concentrations tested (0.89 and 0.9 average 1/k at 4 and mg/l respectively) (p > 0.05) . Fungicidal effects were observed with caspofungin above 0.25 mg/l in the case of six isolates, except for isolates 21,605 and 41,785. It is noteworthy that a pronounced “mini-paradoxical” effect was observed for these two isolates in response to caspofungin exposure (k was 1.23 vs. 0.18 respectively), showing concentration-dependent activity; however, this effect was strongly isolate-dependent. Growth was observed at 0.25 mg/l in the cases of isolates 41,852 and 21,605, while a weak fungistatic effect was detected against six of the eight strains. Surprisingly, 2 mg/l was not fungicidal against isolates 41,852 and 42,268 after 24 h of exposure, while 1 mg/l exerted the fungicidal endpoint in the case of the other six isolates. The average time necessary to achieve the fungicidal endpoint for amphotericin B at 2 mg/l was similar to anidulafungin at 8 mg/l. As expected, fluconazole had a fungistatic activity between 2 mg/l and 8 mg/l against all isolates and achieved a 50% CFU decrease against only two of the eight isolates  at the highest tested concentration (8 mg/l).Two mg/l of amphotericin B exerted a higher killing rate compared to 0.25 mg/l  proved to be as good biofilm formers compared to the SC5314 C. albicans reference strain in our experimental setting . Overall, micafungin showed the highest anti-biofilm effect with a median biofilm MIC ranging from 0.125 to 0.5 mg/l. In contrast, the two other echinocandins produced a weaker efficacy, where median sessile MICs ranged from 0.25 to 2 mg/l and from 0.06 to 2 mg/l for caspofungin and anidulafungin, respectively. Fluconazole had a negligible effect on sessile populations with an average median sessile MIC of > 512 mg/l. In the case of fluconazole, a 90% decrease in metabolic activity was not achieved after 24 h for any of the isolates at the tested concentration ranges  [Cy. fabianii, even though they had susceptible phenotype by standard susceptibility testing. While the biofilm-forming ability of Cy. fabianii isolates presumably played a pivotal role in the reported clinical cases, there are no data on the activity of antifungals against biofilms formed by this species to date. Based on several epidemiological studies, the biofilm formation is associated with higher mortality rates in cases of other rare Candida species [C. albicans reference strain. The activity of the tested antifungals against sessile populations was more diverse compared to the planktonic populations. Based on the obtained MIC values, echinocandins and amphotericin B had comparable activity and that similar to the sessile susceptibility profile of C. kefyr [Cy. fabianii cells is in line with previous studies suggesting that fluconazole is not a biofilm active agent against other Candida spp. [Cy. fabianii; nonetheless, a similar mechanism is implicated in the case of more common Candida isolates [Over the past few decades, the prevalence of infections caused by rare increase , 20. Altreported , 2. Baseregimens , 5. Thisk et al.  Time-kiloperties . Amphoteida spp. . The lacctively) , 14. The species , 24. Of C. kefyr . In contida spp. . Whetherisolates .Cy. fabianii. Our results suggest that echinocandins are a reasonable first-line agents against this species. The activity of amphotericin B against planktonic and sessile populations was also high, but the associated toxicity with the conventional amphotericin B-deoxycholate and the high cost of newer formulations may limit its therapeutic usage for the adult population. In the future, further comparative in vivo experiments are needed to confirm whether these in vitro results have clinical relevance in the treatment of Cy. fabianii infections.To the best of our knowledge, this is the first comprehensive study on the in vitro pharmacodynamic properties of the most frequently used antifungal agents against