The Siglec-Fc protein, a fusion protein combining Siglec with the Fc part of a human antibody, is a promising sialic acid-Siglec axis-targeted agent for cancer treatment and is widely used for Siglec ligands discovery. The recombinant Siglec-Fc fusion protein has been expressed in different cell systems. However, its characteristics have not been investigated in detail. In this study, HEK293 and CHO cell lines were used to express the Siglec9-Fc protein, and their adaptability for production was compared. We optimized culture conditions and compared the glycosylation, yield, dimerization and sialic acid binding activity of the Siglec9-Fc protein produced in HEK293 and CHO. Using purified recombinant protein, we further analyzed the distribution of Siglec9 ligands on cancer cell lines, as well as bladder cancer tissue, and revealed the potential ligands. Our findings provide support for the selection of Siglec9-Fc protein expression systems and detection of related Siglec9 ligands.The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of immunomodulatory receptors expressed in hematopoietic lineages . In humaHypersialylation has been observed in various types of cancer, likely resulting from the elevated metabolic flux of sialic acid as well as the aberrant expression of sialyltransferases and sialidases ,4. Cancein vivo, and interacts with Fc receptors expressed in immune cells such as the NK cell and the macrophage to assist the clearance of “foreigners” and maintain homeostasis [E. coli. So at present, many mammalian cell protein expression systems have been used to produce Siglec-Fc proteins  and the antibody Fc domain mediated biological functions such as antibody-dependent cellular cytotoxicity (ADCC). The sialic acid binding activity is mediated by the V-set domain of the Siglec, and the conserved arginine amino acid is critical for such activity ,10. The eostasis . Therefoeostasis . Additioeostasis . For receostasis ,15. It heostasis . Due to .g., CHO , Expi293.g., CHO ). An extIn this study, we expressed Siglec9-Fc, a representative Siglec-Fc protein, in HEK293 and CHO cell lines, and optimized culture conditions to increase protein yield. We compared glycosylation, sialic acid binding activity and dimerization of the Siglec9-Fc, and further detected ligands distribution on cancer cell lines of different tissue origin and bladder cancer tissue using produced protein. Our findings demonstrated the differences in glycosylation modifications and other protein properties of Siglec9-Fc produced in distinct systems. These results provide experimental support for choosing the appropriate expression system for Siglec-Fc proteins and detecting potential ligands.(1–348)-Fc-6His was ligated into a lentiviral vector pLVX-AcGFP-N1  using ligation mix . Plasmid was amplified using E.coli (DH5α) cultured in an LB medium containing ampicillin. The sequence was confirmed by PCR and DNA Sanger sequencing.The extracellular domain of Siglec9 (1–348) was cloned from human peripheral blood cell (PBMC) cDNA which was kindly provided by Dr. Zhou and the IgG1-Fc domain (99–330) was cloned from pME18Sf-IgLD plasmid, which was kindly provided by Prof. Morihisa Fujita. These two sequences were assembled using Multi-quick cloning kits  and a 6×His tag was linked at the C terminal of the Fc domain for protein purification. Subsequently, the Siglec9v/v) FBS  and 1% (v/v) penicillin/streptomycin  at 37 °C in 5% CO2 atmosphere. T24 (bladder cancer), Jurkat T (lymphocyte leukemia) and CHO (Chinese hamster ovary cell) were cultured in the RPMI 1640 medium  with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37 °C in 5% CO2 atmosphere. All the cell lines were obtained from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences. The lentivirus infection cell lines were storied in our laboratory.HEK293 (human embryonic kidney 293 cell), HEK293T, A549 (lung cancer), K562 (myeloid leukemia), HepG2 (liver cancer), Hela , and MCF7 (breast cancer) were cultured in the DMEM medium  with 10% ) and 10 µL lipofectamine 8000 (Beyotime) were mixed with 0.25 mL Opti-MEM culture medium , respectively. The liquid was blended with plasmids or Lipofectamine 8000 and allowed to rest for 20 min before being added to the HEK293T cells. The cell culture medium was changed after 12 h and the cells were cultured for an additional 48 h. Wild-type HEK293 or CHO cells were incubated with the collected lentivirus medium for 24 h. Cells were placed at 32 °C in a 5% CO2 incubator during the lentiviral package and infection process. Stable transfected cells were selected using puromycin  at the concentration of 1 µg/mL for HEK293 cells and 10 µg/mL for CHO cells.10For the optimization of culture conditions, we set different culture condition combinations , culture temperature  and culture time ), and the product was determined by dot blot. Five independent experiments were conducted on each group of samples.6 stable transfected HEK293 or CHO cells were seeded in a 15 cm cell culture dish. After reaching confluence, cell supernatant was harvested 5 days later and filtered through a 0.22 µm filter. The purified protein was obtained by Ni affinity chromatography  according to the manufacturer’s instructions. The purified protein was then dissolved in PBS containing 1% glycerol and stored at −20 °C.3 × 106 cells were harvested using collected buffer ) and separated into two groups. One group was incubated with 15 units/mL of sialidase  for 30 min at 37 °C. After sialidase treatment, stained cells with purified Siglec9-Fc (7 μg/mL) and PE-conjugated goat anti-human IgG Fc  for 30 min. The cells were then washed with FACS buffer (PBS containing 1% BSA and 0.1% NaN3) and the sample was analyzed using Accuri C6 instrument . Data were further processed using FlowJo (v10.8.1) software.10For protein dimerization analysis, 2 µg of purified protein was mixed with SDS loading buffer with or without β-mercaptoethanol (ME). The sample with ME was boiled for 5 min while the sample without ME was denatured at 37 °C for 1 h. Then, samples were sent to western blot analysis. For protein glycosylation, 2 µg of purified protein was first reduced with 10 mM of dithiothreitol (DTT) for 45 min at 56 °C, then alkylated with 20 mM iodoacetamide (IAM) for 30 min in the dark at room temperature. The protein was digested with or without 0.5 µg PNGase F  for 12 h at 37 °C. The SDS loading buffer was added to samples and the western blot was used to analyze the changes in the molecular weight of the protein.Protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane . The dot blot was performed as previously described . In brieThe method for tissue immunofluorescence was described previously . In brieFor cell immunofluorescence, cells were seeded onto glass coverslips, followed by a PBS wash and fixation with 4% para-formaldehyde for 10 min at room temperature. The subsequent blocking and staining steps were the same as those for tissue immunofluorescence. For cell immunofluorescence, cell actin was stained using Actin-Tracker Green (Beyotime).Images were captured with a Nikon C2si confocal microscope using a CFI Plan Apochromat VC oil objective lens and adjusted with ImageJ  software.w/w), followed by incubation overnight at 37 °C. The peptides were desalted using C18 resin pipet tips .T24 cells cultured in a 10 cm dish were harvested and lysed with 1 mL RIPA buffer (Beyotime) containing 1% protease inhibitor  on ice for 30 min. The supernatant was obtained by centrifugation and divided into treatment and control groups. 50 µL of prewashed protein A&G beads (Beyotime) and 20 µg HEK293-produced and purified Siglec9-Fc were added to the treatment group, and an equal amount of protein A & G beads were added to the control group. After mixing at 4 °C for 2 h, the beads were washed with PBS and eluted with 0.2 M glycine-HCl buffer (pH = 3.0) to get proteins; the pH of the solution was then adjusted to 7.0 using 1 M Tris-HCl buffer (pH = 8.5). IP proteins or 20 µg purified Siglec9-Fc were reduced using 10 mM DTT at 56 °C for 45 min and alkylated with 20 mM IAM in the dark for 30 min at room temperature. Sequencing grade trypsin  was added to the solution at 1:40 (5 and maximum injection time 250 ms) were collected from 350 to 2000 m/z at a resolution of 60,000, followed by data-dependent HCD MS/MS .Lyophilized peptides were suspended in 2% ACN/0.1% formic acid (FA) solution. The samples were first separated on an EASY-nLC 1200 system  with an RSLC C18 column (1.9 µm × 100 µm × 20 cm) packed in house and then coupled with a high-resolution Orbitrap Fusion Lumos spectrometer . The mobile phase consisted of water (A), 0.1% FA and 90% ACN (B), and the gradient elution was 2–4% B, 8 min; 4–20% B, 80 min, 20–32% B, 22 min; 32–80% B, 5 min; and 80% B, 5 min. The flow rate was maintained at 500 nL/min. Spectra  cell or opposing (trans) cell [BSG, ITGB1, CD44 genes were identified, and they may interact with Siglec9  cell . This ma Siglec9 . These pOur results suggest that CHO and HEK293 are suitable for producing Siglec9-Fc, and CHO is better than HEK293 for producing a higher ADCC activity and longer half-life antibodies. Moreover, our study provides experimental evidence for the selection of Siglec-Fc protein expression systems as well as for related Siglec ligands’ detection.


Although the CSE mutation of M. loti did not affect the early stages of symbiosis, i.e., infection and nodulation, with Lotus japonicus, it reduced the nitrogenase activity of nodules and induced their early senescence. Additionally, changes in the production of sulfur compounds and an increase in reactive oxygen species (ROS) were observed in the infected cells of nodules induced by the cse mutants. The effects of CSE inhibitors in the L. japonicus rhizosphere on symbiosis with M. loti were also investigated. All three CSE inhibitors suppressed infection and nodulation by M. loti concomitant with decreased RSS levels and increased ROS and nitric oxide levels. Therefore, RSS derived from the CSE activity of both the microsymbiont and host plant are required for symbiosis, but function at different stages of symbiosis, possibly with crosstalk with other reactive molecular species.Reactive sulfur species (RSS) are present in root nodules; however, their role in symbiosis and the mechanisms underlying their production remain unclear. We herein investigated whether RSS produced by the cystathionine γ-lyase (CSE) of microsymbionts are involved in root nodule symbiosis. A  Various nutrient molecules, including sulfur, are involved in establishing this symbiosis. In pea (Pisum sativum L.) grown in sulfur-deficient soil, nitrogen-fixation activity, plant growth, and yield were reduced, and these phenotypes were improved by supplying large amounts of sulfur (Trifolium repens), sulfur deficiency was shown to affect nodule numbers, nitrogen-fixation activity, and amino acid metabolism in the nodules , including sulfane sulfur and hydrogen sulfide (H2S), function as signal molecules and reductants in vivo  in the root hair and infected cells in nodules, and its crosstalk with H2S, reactive oxygen species (ROS), and reactive nitrogen species (RNS) is considered to be involved in the establishment of symbiosis , cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfur transferase  of M. loti strain MAFF303099 (–2 s–1 (16-h photoperiod) at 25°C for 4, 6, and 8 weeks post-inoculation (wpi). In MAFF303099, the CSE gene is encoded on two open reading frames, mlr1566 and mll4503. Among them, mll4503 gas been predicted by MicrobesOnline (http://www.microbesonline.org/operons/gnc266835.html) to be on the same operon as CBS (mll4505), which functions cooperatively with CSE in RSS production. Therefore, in this study, we focused on the CSE encoded by mll4503, which is predicted to be closely involved in the production of RSS as a “CSE gene”. STM strains of M. loti carry a Tn5-based mini-transposon, randomly distributed throughout the genome  of the culture was 0.4–0.5, cells were harvested, washed twice with yeast mannitol (YM) medium (600 of 0.2. Hydrogen peroxide (H2O2) was used to elicit oxidative stress; it was added to the bacterial suspension to produce final concentrations of 3, 6, and 9 mM, and the suspensions were incubated with shaking at 26°C for 24 h. Sodium cyanide (NaCN) as a sulfane sulfur scavenger was added to the bacterial suspension to produce final concentrations of 5, 10, 50, and 100 μM, and the suspensions were incubated with shaking at 26°C for 16 h. DL-propargylglycine (PAG), β-cyanoalanine (BCA), D-penicillamine (D-pen) (as CSE inhibitors), and hypotaurine (HT) (as a H2S scavenger) were added to bacterial suspensions to produce final concentrations of 100 μM, and the suspensions were incubated with shaking at 26°C for 16 h. OD600 was measured using Miniphoto518R (Taitec).When the optical density at 600 nm (OD) medium , and sus2H2:air=1:9 [v/v]). After a 1-h incubation at 25°C, the level of ethylene in the gas phase was evaluated using a GC-8A gas chromatograph (Shimadzu).The nitrogenase activity of nodules was assessed by measuring acetylene reduction activity (ARA) according to the method reported by To create agar sections of nodules, nodules were embedded in 5% agar and sectioned to a thickness of 70 μm using a vibratome. To create resin sections of nodules, nodules were fixed with 4.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) at 4°C overnight. Fixed samples were then dehydrated using a graded ethanol series, embedded in JB4 resin (Polysciences), and sectioned to a thickness of 3 μm. Nodule sections were stained with 0.5% (w/v) toluidine blue reagent according to the manufacturer’s instructions.2HPO4, and 2 mM NaH2PO4 (pH 7.4). To detect sulfane sulfur, the roots and nodules were soaked in 10 μM SSP4 (Dojindo) with 0.5 mM cetyltrimethylammonium bromide. SSP4 has no membrane permeability, but may penetrate cell membranes in combination with cetyltrimethylammonium bromide. SSP4 reacts with sulfane sulfur and with the polysulfides of glutathione and cysteine. To detect NO, samples were soaked in 20 μM DAF-FM DA . DAF-FM DA has membrane permeability and is deacetylated to DAF-FM by esterase inside the cell, wherein DAF-FM reacts with the endogenous NO oxidation product N2O3 to form a highly fluorescent triazole. To detect ROS, samples were soaked in 10 μM CellROX Green Reagent (Invitrogen). The cell-permeable CellROX reagent is essentially non-fluorescent in its reduced state, but exhibits a strong fluorescent signal upon oxidation. To detect H2S, samples were soaked in 5 μM Hsip-1 DA (Dojindo). Hsip-1 DA has membrane permeability. Through esterase activity inside the cell, Hsip-1 DA is deacetylated to Hsip-1, which is a fluorescent molecule (a copper [II] ion chelate type) that specifically reacts with H2S. In the roots, the endogenous production of each signal molecule was detected via fluorescence or stereofluorescence microscopy. Fluorescence images were captured using an A1si-90i microscope and epifluorescence images with an Eclipse 90i microscope (both from Nikon). In all cases, fluorescence intensity was quantified using Image J .The production of signal molecules in roots and nodules was detected and measured as previously described .Sulfane sulfur, H13C15N L-valine). The supernatant was collected after centrifugation at 13,000 rpm and 4°C for 3 min and then evaporated in a centrifugal concentrator. The residue was dissolved in 20 μL of 0.1% formic acid and subjected to a LC–MS/MS analysis using a Triple TOF 5600 system (AB Sciex).Freshly collected samples were frozen and stored at –80°C until used. A sulfur metabolomic analysis was performed by the Sulfur Index service in Japan using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) as previously described (2S under free-living conditions was compared between wild-type (WT) M. loti strain MAFF303099 .At 8 wpi, plants inoculated with  with WT A and B. cse mutants were examined histologically to investigate nodule senescence. At 5 wpi , many infected cells remained normal in the nodules of plants inoculated with WT, whereas infected cells were partially disintegrated in the nodules of plants inoculated with the cse mutants  was counted 10 days after the inoculation using DsRed-labeled M. loti, and the number of nodules was counted at 4 wpi. PAG, BCA, D-pen, NaCN, and HT each reduced the number of ITs and nodules . None of the inhibitors or scavengers significantly affected nitrogenase activity C.L. japonicus were treated with the CSE inhibitors and scavengers to evaluate their effects on sulfane sulfur and H2S levels in the roots. Surface-sterilized seeds were placed on an agar plate supplemented with each inhibitor or scavenger. Three days later, the seedlings were treated with SSP4 and HSip-1 DA, and sulfane sulfur and H2S levels were estimated according to fluorescence intensity. Each of the inhibitors and scavengers, except for NaCN, reduced the fluorescence intensity of SSP4 , indicating that the early stages of symbiosis were significantly affected. The increased levels of ROS and RNS, which function negatively in the early stages of symbiosis when present at excessive levels, suggest that crosstalk among reactive molecular species inhibits infection. However, none of the inhibitors or scavengers examined inhibited the nitrogenase activity of L. japonicus nodules under the experimental conditions used in the present study  Reactive Sulfur Species Produced by Cystathionine γ-lyase Function in the Establishment of https://doi.org/10.1264/jsme2.ME23021Supplementary Material


Salmonella enterica, with potential application in poultry feed for biocontrol of that pathogen. This pathogen is known as one of the main microorganisms responsible for contamination in the food industry and in foodstuff. The current techniques for decontamination and pathogen control in the food industry can be very expensive, not very selective, and even outdated, such as the use of broad-spectrum antibiotics that end up selecting resistant bacteria. Hence, there is a need for new technologies for pathogen biocontrol. In this context, bacteriophage-based biocontrol appears as a potential alternative. As a cocktail, both phages were able to significantly reduce the bacterial load after 12 h of treatment, at either multiplicity of infection (MOI) 1 and 10, by 84.3% and 87.6%, respectively. Entrapment of the phage virions within the EBMW matrix did not exert any deleterious effect upon their lytic activity. The results obtained showed high promise for integration in poultry feed aiming at controlling Salmonella enterica, since the edible biopolymeric microcapsular wrapping integrating lytic bacteriophage particles developed was successful in maintaining lytic phage viability while fully stabilizing the phage particles.This research work aimed at developing an edible biopolymeric microcapsular wrapping (EBMW) integrating lytic bacteriophage particles for  Salmonella enterica, Escherichia coli, and Staphylococcus aureus [Foodborne diseases are a major cause of morbidity and mortality worldwide. According to the World Health Organization (WHO), it is estimated that diarrheal diseases alone (most of which are caused by foodstuff contaminated by pathogenic microorganisms) kill 1.9 million children per year . In Brazs aureus ,4, causiSalmonella is considered a member of the Gram-negative Enterobacteriaceae family [Salmonella enterica and Salmonella bongori, and has over 2500 known serotypes that differ in their wide host range and ability to cause disease, with over 50% of the identified serotypes belonging to the Salmonella enterica species, which is responsible for the vast majority of Salmonella infections in humans [Salmonella is part of the microbiota of birds, and there is a diverse amount of Salmonella serovars. While the serovars Salmonella pullorum and Salmonella gallinarum are important pathogens of birds, the serovars Salmonella enteritidis and Salmonella typhimurium have a broad host spectrum [Salmonella alone is responsible for several tens of millions of incidents of salmonellosis worldwide, on a yearly basis, associated with more than 150,000 deaths [Discovered and described in 1885 by Daniel Salmon, a veterinary bacteriologist, the genus n humans . Salmonespectrum . Among o0 deaths ,5.From the second half of the twentieth century, the emergence of bacterial strains resistant to multiple drugs (viz. multi-drug resistant) has been a reality arising from broad, indiscriminate utilization of chemical antibiotics in areas as diverse as (but not limited to) human medicine, animal medicine, food industry, and agriculture ,8. In adPoultry production is one of the world’s sectors that most uses antibiotics, and reducing the use of antibiotics is one of the biggest challenges for this industry globally . In thisBacteriophages are viruses devoid of metabolic machinery of their own that exclusively infect susceptible bacterial cells, hence being obligate intracellular parasites that require a bacterial host cell to replicate ,14. The Salmonella spp. by the application of bacteriophages in chickens and products derived from them. Additionally, bacteriophage supplementation has been shown to improve feed efficiency, reduce pathogens in broilers, and improve production and egg quality in laying hens [In the poultry industry, bacteriophages have also been used in a wide variety of applications, such as treating live birds, adding to poultry products, and disinfecting processing equipment. It was shown that treatment with phages administered to chickens via aerosol or oral gavage was able to control bacterial infections and decrease mortality . Recent ing hens .For the success of antibacterial therapy with phages, several obstacles still have to be overcome, with one of the problems of phage therapy for birds  being the form of administration. In this sense, the incorporation of phage particles into poultry feeds could be an interesting alternative. For this, the phage must be able to survive in the feed and the gastrointestinal tract of the birds. The acidic environment of the stomach can promote the deactivation of phage particles . AbioticSalmonella enterica and promote their structural and functional stabilization within biopolymeric microcapsular wrappings aiming at potential applications in poultry feed for the biocontrol of Salmonella enterica in live poultry and the associated foodstuff thereof (eggs and carcasses).With all the aforementioned facts in mind, the major goal of the research work entertained herein was to isolate and characterize lytic bacteriophage particles for Bacterial host for phage isolation: The collection Salmonella enterica CCCD-S004 strain utilized in this work as host for phage isolation was acquired from CEFAR Diagnóstica . Bacteriophages: The two phages utilized in this study (ph001L and ph001T) were previously isolated from samples of lake water (ph001L) and soil with hen faeces (ph001T) collected near the Veterinary Hospital at UNISO , Sorocaba/SP (Brazil). Collection strains for host-range assays: The bacterial strains utilized in the extended host-range assays were obtained either from ATCC  , CEFAR  , IBSBF  (viz. Pseudomonas syringae pv. garcae IBSBF-158), and NCTC , Salisbury, UK) (viz. Klebsiella pneumoniae NCTC-13439). All manipulation of materials, bacteria, bacteriophages, culture media, and evaluation of microbiological activity was carried out in a Filterflux® Class II, type B2, biological safety cabinet model SP-SBIIB2-126 from SP-LABOR .−1 in a Master System All MS2000 .The chemicals utilized in this study were purchased from Dinâmica Química Contemporânea Ltd.a . Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) culture media were purchased from Sigma-Aldrich Brazil , and bacteriologic solid agar was purchased from Gibco Diagnostics . Sterilizing filtration systems Stericup™-GP (with 0.22 µm pore diameter polyethersulphate membrane) were acquired from Merck-Millipore . Tap water was ultra-purified to a final resistivity of 18.18 MΩ·cm and conductivity of 0.05 µS·cmThe host bacteria  was hydrated in TSB liquid medium, plated on solid TSA, and incubated at 37 °C for 12 h. A single CFU was then withdrawn with a sterile loop, inoculated in 250 mL sterile TSB, and incubated at 37 °C for 12 h. At predetermined time intervals, the optical density of the culture was evaluated spectrophotometrically at 610 nm.Salmonella enterica CCCD-S004 .Phage enrichment from samples of lake water and soil with hen faeces collected near the Veterinary Hospital at UNISO was performed according to the procedure described elsewhere ,24,25, wIsolation of phage plaques was carried out using the conventional double-layer agar method described in previous works ,24,25,26Salmonella enterica overnight culture (100 µL) was mixed with 5 mL of molten top agar–TSB  in test tubes, tapped gently, and poured onto TSA plates which were gently swirled and allowed to dry out for 1–2 min, followed by overnight incubation at 37 °C. Sterile paper strips were wetted in the phage enrichment suspension and dragged several times on a Petri plate containing a bacterial lawn. The plate was incubated at 37 °C for 24 h and then the morphology of the phage plaques was observed. Different phage plaques were pierced with sterile toothpicks, which were then stuck several times (in a line) in Petri plates with bacterial lawn. Sterile paper strips were then used to streak the phages as described above. More successive single-plaque isolation cycles were performed to obtain pure phage isolates. The plates with different plaque morphologies were then incubated overnight at 37 °C, and the last two steps were repeated until all phage plaques were uniform. The plates were stored at 4 °C until needed. To each plate used, 5 mL of SM buffer was added, and the plates were then further incubated with shaking (70 rpm) at 4 °C for 18 h. After incubation, the SM buffer with phages was collected and added to chloroform up to a final ratio of 10% (v/v). The phage suspensions were centrifuged  to remove intact bacteria or bacterial debris. The aqueous phases were collected into a single sterile flask and stored at 4 °C as phage stocks.A Bacteriophage enumeration was carried out via the double agar overlay technique, as follows. Serial dilutions of the concentrated stock bacteriophage suspensions produced were prepared sequentially, starting by adding 50 µL of each concentrated stock bacteriophage suspension to 450 µL SM buffer. An amount of 5 µL-droplets of each bacteriophage dilution were plated in triplicate in a lawn of the bacterial host and the plates were allowed to dry out for 10 min, after which they were incubated overnight at 37 °C. Following incubation, the bacteriophage plaques formed in each serial dilution were counted, considering only those dilutions with 3–30 bacteriophage plaques. The bacteriophage titre (PFU/mL) of the concentrated stock bacteriophage suspensions was then calculated as 10–1011 PFU/mL) were added to a sterile mixture of polyethylene glycol (PEG) 8000   and NaCl (1 M) , in a volumetric proportion of 2:1, respectively. The resulting suspensions were incubated overnight at 4 °C and then centrifuged at 11,000 rpm . The supernatant was then discarded, and the pellet was resuspended and homogenized in a 5 mM MgSO4 aqueous solution .Phage suspensions  in a benchtop Beckman-Coulter ultracentrifuge (model Optima TLX micro-ultracentrifuge) with a TLA-55 Fixed-Angle Rotor  and underwent negative staining with uranyl acetate  at 2% (w/v) and pH 7.0, following the procedure described in previous works [6 filament, operating at 200 kV and with resolution of 0.23 nm; a high-resolution CCD camera from GATAN Inc.  with a resolution of 11 Mp (4.0 × 2.7 k·pixels/9 × 9 µm2) was utilized for the acquisition of digital images, via the software Gatan Microscopy Suite .Phage particles of PEG-concentrated suspensions were centrifuged  from the National Institute of Health .Salmonella enterica CCCD-S004 , as Phage host-range was determined by spot-testing using the bacterial strains listed in Table 4, according to the procedure described elsewhere ,28,29. Feria×100 ,28,30,31Salmonella enterica CCCD-S004 (1 × 108 CFU/mL) and ph001L (1 × 105 PFU/mL) and ph001T (1 × 105 PFU/mL) at MOI ≤ 0.001 [tP, P0, and P∞ are phage concentrations (PFU/mL) at times t, 0, and ∞, respectively, t is the incubation time (min). The model was fitted to the experimental phage growth data via nonlinear regression analysis using the function “Solver” within Microsoft Excel .Growth parameters for the two phages were extracted from the one-step growth curves using  ≤ 0.001 ,25, withny yield ,24,25,29Salmonella enterica CCCD-S004 suspension in exponential growth  was added to bacteriophage particles at 8 5 CFU/mL in order to produce MOI 0.001 [tP and P0 are phage concentrations (PFU·mL−1) at times t and 0, respectively, δ is the (first order) phage virion adsorption rate onto susceptible bacteria (CFU−1·mL·min−1), ϕ is the (first order) phage virion desorption rate from reversible virion–bacteria complexes (mL·min−1), 0X is the initial concentration of uninfected (but susceptible) bacteria (CFU/mL), and t is infection time (h). The model was then fitted to the experimental phage adsorption data via nonlinear regression analysis using the function “Solver” within Microsoft Excel , allowing determination of the phage adsorption rate.A OI 0.001 , so thatOI 0.001 ,24,25. POI 0.001 ,34,35, v5 CFU/mL, exponential growth) by the phages was studied at MOI values 0.01, 0.1, 1, 10, 100, and 1000. For each MOI experiment (performed in triplicate in three independent assays), a bacterial control (BC) was also added, comprising only planktonic host cells. Both BC and treatments  were incubated with the same time/temperature parameters, viz. 37 °C and 12 h. Two mL-aliquots of BC and treatment samples (BP-B) were withdrawn at predetermined intervals of time up to a total treatment timeframe of 12 h, viz. 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 420, 480, 540, 600, 660, and 720 min, and their absorbance duly measured at a wavelength of 610 nm.Inactivation of planktonic host cells (107–8 PFU/mL) viability was studied in phosphate-buffered saline . For the pH and T experiments, aliquots were withdrawn every 2 h during the first 12 h and then every 12 h up to 72 h of incubation. For the solar radiation experiments, aliquots were withdrawn every 1 h up to 7 h of exposure to direct sunlight. Phage concentrations were evaluated in triplicate in three independent experiments through double-layer agar plating followed by overnight incubation at 37 °C.The aftermath of T, pH, and solar radiation on phage , under constant temperature (25 °C).The aftermath of T on phages ph001L and ph001T viability was studied via addition of phage suspensions to sterile PBS (pH 7.0), followed by incubation at a constant temperature .2 ). Solar radiation  is the most important factor for the loss of phage infectivity in the environment [The aftermath of solar radiation on phages ph001L and ph001T viability was studied via addition of phage suspensions to sterile PBS (pH 7.0), followed by exposure to natural sunlight . Control samples (SR-C) were not exposed to solar radiation. These studies were carried out on a shiny day with ambient T varying from 29 °C (09h00) to 35 °C (end of the experiment), with a solar irradiance of ca. 4.561 kWh/mironment . Short rironment . In the 7 CFU·mL−1. Hence, the process (w/w) of the total mass of ultrapure water containing the phage cocktail. Occasionally, these dispersions were stirred and allowed to stand by for at least 1 h, to allow complete hydration of the sodium alginate. For the EBMW formulation, 5.0 mL gelatin solution at a concentration of 10 mg/mL and 5 mL of the phage cocktail at a given MOI were thoroughly mixed together in a beaker and, afterwards, the resulting suspension was added to 20 mL of sodium alginate at 2% (w/w). The suspension thus produced   the phage cocktail followed the internal gelation procedure described elsewhere ,37,38, w process  was initTo check the preservation of lytic viability of the bacteriophage particles entrapped within EBMW, a sample of the formulation was placed on a bacterial lawn of the host bacteria on a Petri plate followed by incubation at 37 °C for 24 h. After this time period, the presence (or not) of clear lysis zones surrounding the EBMW sample was observed. host cells  by the EBMW particles with entrapped phage cocktail at MOI values 100 and 1000 was studied. For each EBMW formulation (performed in triplicate in three independent assays), a bacterial control (BC) was also prepared, comprising only planktonic host cells. Both BC and treatments  were incubated with the same time/temperature parameters, viz. 37 °C and 12 h. A total of 10 microparticles of a given formulation were placed in a 0.5 mL conical-bottom bioreactor containing a Teflon-coated conical stirring bar. An amount of 500 microlitres of bacterial suspension  was then added, magnetic stirring (75 rpm) was initiated, and the bioreactor was placed in an incubation chamber set at 37 °C. Five μL-aliquots of BC and treatment samples (EBMW-BP-B) were withdrawn at predetermined intervals of time up to a total treatment timeframe of 12 h, viz. 0, 15, 30, 45, 60, 120, 240, 360, 480, and 720 min, and serial diluted in 45 μL SM buffer. The bacterial concentration in both BC and EBMW-BP-B dilutions was determined in triplicate in solid TSA medium via the drop (5 μL)-plate method after an incubation period of 12 h at 37 °C.Inactivation of planktonic Salmonella enterica bacterial lawn , after an incubation period of 12 h at 37 °C.To verify the preservation of lytic activity of the entrapped phage particles and to assess their release from the EBMW particles, a simple experiment was performed. A total of 10 EBMW microparticles with entrapped phages at MOI 1000 were placed in a 0.5 mL conical-bottom bioreactor containing a Teflon-coated conical stirring bar. An amount of 500 microlitres of SM buffer was then added, magnetic stirring (75 rpm) was initiated, and the bioreactor was left at room temperature ca. 25 °C). Five µL-aliquots of the supernatant were withdrawn at predetermined intervals of time up to a total timeframe of 3 h, viz. 0, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min, and serial diluted in 45 µL SM buffer. The phage titre in all dilutions was determined in triplicate by the double agar-layer method ,24,25, u °C. FiveThe physicochemical characterization of the EBMW formulations  involved−1 to 675 cm−1 , with Happ–Genzel apodization.FTIR spectra of EBMW formulations 1 and 5 were obtained in a FTIR spectrophotometer from Thermo Scientific  coupled with an ATR module  (Smart Omni Sampler), from 4000 cm−1, under a constant N2 flow of 50 mL·min−1, with the heat absorbed by the samples being recorded at a sampling rate of 0.2 s per data point.Thermal analyses (DSC) of EBMW formulations  were performed in a microcalorimeter from METTLER TOLEDO , according to Rocha et al. , using h2 silicon drift detector (SDD) operating at Patm. All tests were performed with a measuring timeframe of 300 s using atmospheric air, and the spectra were obtained sequentially from 0 keV to 30 keV (resolution of 0.02 keV).The elemental makeup of EBMW formulations (plain and loaded with bacteriophage cocktail at MOI 1000) was determined using an EDXRF spectrometer  equipped with a 5 W, 10–50 kV, Ag anode X-ray tube, with energy resolution of 125 eV, filters of Ag, Cu, Ti, and Al for the X-ray beams, and a high-resolution 25 mmTomographic images of the EBMW formulation entrapping the phage cocktail at MOI 1000 were obtained in an X-ray transmission tomograph  from BruThe surface and morphology of an EBMW particle were analyzed in a SEM  at high-vacuum. The samples were sputter-coated with a 92 Å-thick Au film via cathodic pulverization, in a metalizing device . Photomicrographs were collected via random scanning using electron beams at acceleration speeds of 10.0 keV.F-ratio (lack of fit mean square (SSlack of fit/NDFlack of fit) over the replications mean square (SSreplications/NDFreplications)) was compared with the statistical F-value ). SS = sum of squares, NDF = number of degrees of freedom.Statistical tests of lack of fit of the mathematical model for phage adsorption  to the experimental phage adsorption data were undertaken, aiming at testing the goodness of the nonlinear fittings. These statistical tests are based on the fact that the subspace containing the experimental data replications is orthogonal to the subspace containing both the experimental data averages and the expectation function ,42,43,44p < 0.05 was considered to be statistically significant.The data gathered in the in vitro phage–bacteria inactivation assays was statistically analyzed with GraphPad Prism 7.04 . While the normal distribution of the experimental inactivation data was verified by a Kolmogorov–Smirnov test, the homoscedasticity was verified by the Levene’s test. The significance of the differences recorded for bacterial concentration was evaluated via comparison between the results of treatment samples for each MOI  with the corresponding bacterial concentrations in the control  for the different inactivation times, using two-way ANOVA and Bonferroni post hoc tests. A value of Salmonella enterica has been proposed, aiming at potential applications in poultry feed for the biocontrol of this pathogen. Two different virulent phages were selected based on their ability to form clear plaques of lysis, which were amplified in a Salmonella enterica CCCD-S004 bacterial strain and, to assess their infectious potential for biotechnological applications such as biocontrol of the aforementioned pathogen in live poultry, physicochemical and biological characterization was undertaken, together with the characterization of the EBMW formulation integrating the lytic cocktail.In the present research work, the formulation of a bioactive edible biopolymeric microcapsular wrapping (EBMW) integrating a structurally and functionally stabilized cocktail of two newly isolated lytic phages for Salmonella enterica CCCD-S004), and therefore the production of a growth curve (9 CFU/mL (corresponding to a maximum absorbance of 1.1049), and of the lag period as 345 min (ca. 5.75 h).For isolating lytic bacteriophage particles, one decided to use a collection bacterium  lawn , produced clear plaques with very small dimensions on the host (04) lawn , with abBased on the morphological analysis entailed by TEM , both phUV-Vis spectral scans of PEG-concentrated phages ph001L and ph001T a were usFor the two phages, maximum absorption was observed around 251 nm (phage ph001L) and 252 nm (phage ph001T), and the minimum absorption that was observed around 245 nm was an indication that bacterial cell debris were virtually absent from the preparation with the concomitant presence of a high concentration of phage virions.251(2) nm − Abs320 nm = ƒ ), allowed obtaining the molar extinction coefficient of the newly isolated phages as εphage ph001L = 6.463 × 10−12 (PFU/mL)−1·cm−1 and εphage ph001T = 9.986 × 10−12 (PFU/mL)−1·cm−1. By subtracting Abs320nm, a wavelength where there is little absorption of radiation from phage chromophores, a raw correction for light scattering from phage particles and non-phage particulate contaminants was carried out [Fitting the Beer–Lambert linear relationship to the experimental data displayed in ried out ,27,28.Phage virions are essentially made of structural proteins such as capsid, tail, baseplate, and spike, whose chromophores  exhibit a maximum absorption of radiation around 278–280 nm in the ultraviolet wavelength region. Hence, quenching of radiation owing to protein chromophore absorption and scattering by whole virion particles corrected by the quenching of radiation owing solely to protein chromophores, leads to the hallmark absorbance for a particular virion at a particular concentration . StructuAccording to previous studies ,25,26,29Salmonella enterica subsp. Enteritidis ATCC 13076, Pseudomonas syringae pv. Garcae IBSBF-158, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC-13883, and Klebsiella pneumoniae NCTC-13439, with moderate efficacies  of 10 min, a latent period (lp) of 25 min, and an intracellular accumulation period (iap) of 15 min, with a virion morphogenesis yield (bs) of 466 virions/host cell, whereas phage ph001T presented an eclipse period (ep) of 10 min, a latent period (lp) of 40 min, and an intracellular accumulation period (iap) of 30 min, with a virion morphogenesis yield (bs) of 132 virions/host cell.Salmonella enterica CCCD-S004 cells after 30 min and 100% adsorbed after 60 min  = 0.96384); δph001T = 7.000 × 10−10 CFU−1·mL·min−1 and φph001T = 4.000 × 10−4 mL·min−1  = 0.96791).Fitting the experimental phage virion adsorption data to the adsorption decay model via nonlinear regression enabled estimation of the phage virion adsorption rates onto their host cells . Because “lack of fit” arises from the oscillation of experimental data points around the model fitted, a p-value > 0.10 , allows one to conclude that the mathematical model fits  the actual response data. Such a conclusion was illustrated via inclusion of small plots in A statistical test of lack of fit of the adsorption decay model depicted in p < 0.05) during the 12 h of incubation (p > 0.05) (p < 0.05), with phage ph001L. At MOI 10, the performance of this phage was similar to at MOI 1 . However, at MOI 100, phage ph001L reduced the bacterial load by 58.0% after 6 h of incubation with the host, which was reduced to only 26.6% after 12 h, due to bacterial regrowth (p > 0.05) (p < 0.05), for independently applied phage ph001L (p > 0.05) of bacterial load reduction after 12 h of incubation with the host. Increasing the MOI from 0.01 → 0.1 → 1 → 10 → 100 → 1000 did significantly increase the inactivation factor after 6 h of incubation , for independently applied phage ph001T (p < 0.05) reducing the bacterial load between 6 h and 12 h of incubation with the host (p > 0.05). When phage ph001L was used, a significant bacterial regrowth was observed after 8 h of incubation  for MOI 1, 10, 100, and 1000 (p > 0.05). Despite this, by the end of the incubation timeframe, the bacterial densities in the different treatments using either independent phages ph001L and ph001T or a cocktail of both phages were significantly lower than that observed for the bacterial control  increased substantially  up to 12 h of incubation, after which phage ph001L viability decreased more at pH 8.0 than at pH 6.5 (p < 0.05). However, at pH 3.0, phage ph001L endured during the first 2 h (p > 0.05) but lost all its lytic viability (p < 0.05) after 4 h. At pH values 6.5 and 8.0, phage ph001L persisted as viable for at least 72 h at 25 °C (p < 0.05). After 72 h of incubation at pH 6.5 the abundance of phage ph001T decreased by about two orders of magnitude (p < 0.05). Nevertheless, the reduction of lytic viability was more significant at pH 6.5 for this phage than at pH 8.0, after 24 h of incubation (p < 0.05). At pH 9.0, phage ph001T lost 1.07 log·PFU/mL after 2 h, 2.35 log·PFU/mL after 6 h, and completely lost its lytic viability after 12 h  were tested, it was observed that phage ph001L concentration decreased with the decrease in pH; however, the differences among pH values 6.5 and 8.0 were not statistically significant (p < 0.05). A maximum decrease of ca. 1 log·PFU/mL was observed after 72 h when the phage ph001L samples were kept at a temperature of 25 °C, a trend that was also observed at 41 °C (p < 0.05). Regarding phage ph001T, it was completely stable at 25 °C (p > 0.05), but at 41 °C lost 0.8 log·PFU/mL after 48 h of incubation and ca. 1.8 log·PFU/mL after 72 h (p < 0.05). At 50 °C, phage ph001T lost ca. 1.6 log·PFU/mL after only 4 h of incubation, but after this timeframe it ceased to be viable (p < 0.05).The reduction in the concentration of viable phage ph001L particles was much higher at 50 °C than at 25 °C and 41 °C (p < 0.05) and 3.8 log·PFU/mL  in the abundance of viable phage virion particles, when compared to the controls (SR-C).Exposure of phages ph001L and ph001T to direct sunlight for 7 h promoted a decrease of 2.2 log·PFU/mL . Lysis zones can be seen in the lawn, surrounding the microcapsular wrappings integrating the phage cocktail at MOI 1 , a putative mechanism was put forward , chitosan becomes deprotonated and acquires a net negative charge, and the repulsion forces acting on the  Ca alginate matrix prevents surface rebinding. The increase in pH promotes a disentanglement of the two polymers, destructuring the particle and promoting release of the phage virions into the outer medium, where the phage virions can contact and infect the bacterial host cells, promoting their lysis, as can be observed in The EBMW matrices integrating the phage cocktail at MOI 100 and MOI 1000 were also used in microscale bacterial inactivation assays, and the results obtained are displayed in As can be observed from inspection of the data in From the simple experiment that was designed and implemented aiming at evaluating the process of release of the phage virions from the EBMW formulation with entrapped phage particles at MOI 1000, it is clear that the EBMW particles released the virions progressively with time and that they retained their lytic activity .−1 corresponds to the elongation of the OH- groups present in both the alginate polymer chain and residual water molecules [−1 and 1635.87/1635.66 cm−1 may be attributed, respectively, to asymmetrical and symmetrical axial distortions of -COO- groupings, indicative of the existence of carboxylic acid residues in the calcium alginate matrix [−1 for the calcium alginate matrix coated with chitosan, arising most likely from isolated alkenes. The existence of N-acetyl moieties originating from chitosan was ascertained by the peaks appearing at ca. 1635 cm−1 (primary amide C=O stretching) and 1295 cm−1 (tertiary amide C-N stretching). No characteristic N-H bondings from secondary amides were found in the spectra of the formulations tested. The peaks at ca. 1420 cm−1 were attributed to bonding of CH2 groups. The small peak at ca. 1144 cm−1 was assigned to asymmetrical stretching of -C-O-C- groups whereas the peaks at 1078 cm−1 and 1028/1029 cm−1 were assigned to stretching vibrations of the -C-O-C- bond of the ether groups from the chitosan coating [−1 was attributed to -C-H- groups bonding out of the plane of the sugar rings in chitosan moieties [−1 was most likely due to stretching of -C-N- bonds from aliphatic amines [−1 was most likely due to antisymmetric stretching of CO2 molecules from air entrapped inadvertently in the EBMW matrices during their formation [The FTIR spectra of the chitosan-coated EBMW integrating or not the phage cocktail are very similar , allowinolecules ,56. The e matrix . Stretch coating ,59,60,61moieties , whereasormation . As can DSC thermograms of a plain EBMW formulation and of an EBMW formulation integrating the phage virion cocktail at MOI 1000 are displayed in 2, and H2 [Salmonella. According to several researchers [Very similar thermal events can be perceived for both EBMW particles, with the sample containing the phage virion cocktail displaying a slightly higher melting enthalpy at virtually the same temperature, viz. 111 °C. The peak temperature of heat absorption of the two particle formulations were very close to one another and virtually equal to the mid-point of the calcium alginate melting range, viz. ca. 111.5 °C . The pre, and H2 . The EBM, and H2 . Phages , and H2 . In addi, and H2  for micrearchers , proteinThe elemental profiles of EBMW formulations, with and without phage particles, are displayed in The most common substances were, as expected, carbon, hydrogen, and oxygen, accounting for ca. 79% and 77% of EBMW formulations without and with the phage cocktail, respectively . CalciumThe optimized EBMW developed may be considered a natural polymer composite exhibiting a very special porous microstructure which enables the imprisonment of the phage particles. From the tomographic analyses via X-ray transmission performed to an EBMW particle loaded with the phage particle cocktail , a homogThe chitosan layer coating the calcium alginate matrix is in greater evidence  { 0.63632 , and thu 0.63632  allow on 0.63632 . AdditioAnalyses of a EBMW particle via SEM allowed one to observe a homogeneous surface without any fissures or crevices .Salmonella enterica is known as one of the main microorganisms responsible for poultry contamination. Developing new alternatives to the conventional antibiotic-based antimicrobial control for preventing and/or controlling infections by this pathogen have been quite challenging and a long-time goal within the scientific community, aiming at reducing the development of multi-drug resistant bacteria. In the research effort described herein, the structural and functional stabilization of two newly isolated lytic phages for S. enterica  within edible biopolymeric microcapsular wrappings (EBMW) has been proposed, aiming at a potential integration in poultry feed as a means to control the aforementioned pathogen. The results obtained in this study provide clear evidence that the use of the two newly isolated phages can reduce the population of pathogenic S. enterica cells. The two newly isolated phages produced translucent and tiny plaques on a lawn of the bacterial host, exhibiting diameters of approximately 0.1 mm . According to Hyman , newly ificacies , yieldinA one step of growth was clearly observed for both phages  during tAdsorption of a free phage virion onto a bacterial host cell is the apotheosis of its existence, with the free energy reserve imparted to the virion three-dimensional conformation during its morphogenesis coming into play, with the bacterial surface receptor-specific adsorption of free phage virions dictating their host range . Hence, −10 CFU−1·mL·min−1 and 7.0 × 10−10 CFU−1·mL·min−1, for phages ph001L and ph001T, respectively  and [−10 mL·min−1 and adsorption rate of phage 2 = 1.8 × 10−10 mL·min−1, onto Salmonella typhi), one order of magnitude lower than results reported by [Salmonella enterica serovar Enteritidis strain S1400, 1.00 × 10−9 mL·CFU−1·PFU−1·h−1) and [Salmonella pullorum SP01, (8.8 ± 0.5) × 10−9 mL·min−1), and two orders of magnitude lower than the results reported by [Salmonella enterica = 1.6 × 10−8 mL·min−1). According to [–8 mL·min−1, but our results  were two orders of magnitude lower than such a maximum. Nevertheless, the desorption rate of phage ph001L (1.900 × 10−3 mL·min−1) was much larger than that of phage ph001T (4.000 × 10−4 mL·min−1), implying that fewer phage ph001L virions endured adsorbed to the host cells. Because adsorption of phage virions onto specific receptors on the host cell followed by the virion genome translocation into the host cytoplasm is required for its effective infection and concomitant virion morphogenesis [Both phages revealed virtually equal adsorption rates onto the host cells, viz. 8.0 × 10ectively . These r−10) and  (adsorptorted by  (phage Ph−1) and   thermal events were observed for both the EBMW formulation devoid of phage particles and the EBMW formulation integrating the cocktail of phage particles , with meThe results from the thermal characterization of the EBMW particles are linked to the stability of the particles, a crucial parameter for their successful incorporation into poultry feeding. The thermal stability of a biopolymer can be defined as its ability to withstand the action of heat while maintaining its properties (such as toughness or elasticity) at a given temperature and as can be observed from inspection of As expected, the EBMW formulations were composed almost entirely of carbon, hydrogen, and oxygen , arisingAnalysis of EBMW containing the cocktail of lytic virions at MOI 1000 by XRT  closely Additionally, morphology analysis of a EBMW particle via SEM allowed one to observe a homogeneous rugged surface (photomicrographs c, d, and e in Salmonella enterica. Yet, both phages could not fully restrain bacterial regrowth in vitro, neither separately nor as a cocktail. Hence, selecting more lytic phages for phage–bacteria inactivation assays should consider not only their efficacy but also their potential for developing phage-resistant bacterial mutants, which should circumvent and virtually obliterate the downside of bacterial-acquired resistance to phages.The results described herein clearly suggest that the cocktail produced with the two newly isolated lytic phages, ph001L and ph001T, have the potential to be an effective surrogate to antibiotics in controlling Salmonella enterica. However, both phages were not able to fully prevent bacterial regrowth, although the bacterial reduction levels were quite high when both phages were used as a cocktail, especially at MOI 1 and 10. It was also demonstrated that microencapsulation within a biopolymeric formulation is a viable method for fully stabilizing the virion particles, both from structural and functional points of view, if integration within poultry feed is sought as a means of controlling Salmonella enterica in poultry.The results of the present work suggest that phage treatment using phages ph001L and ph001T can be an effective alternative to control


Kawasaki disease (KD) is a systemic vasculitis of unknown etiology affecting mainly children. Studies have shown that the pathogenesis of KD may be related to autophagy. Using bioinformatics analysis, we assessed the significance of autophagy-related genes (ARGs) in KD.Common ARGs were identified from the GeneCards Database, the Molecular Signatures Database (MSigDB), and the Gene Expression Omnibus (GEO) database. ARGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein–protein interaction (PPI) network analysis. Furthermore, related microRNAs (miRNAs), transcription factors (TFs), and drug interaction network were predicted. The immune cell infiltration of ARGs in tissues was explored. Finally, we used receiver operating characteristic (ROC) curves and quantitative real-time PCR (qRT-PCR) to validate the diagnostic value and expression levels of ARGs in KD.There were 20 ARGs in total. GO analysis showed that ARGs were mainly rich in autophagy, macro-autophagy, and GTPase activity. KEGG analysis showed that ARGs were mainly rich in autophagy—animal and the collecting duct acid secretion pathway. The expression of WIPI1, WDFY3, ATP6V0E2, RALB, ATP6V1C1, GBA, C9orf72, LRRK2, GNAI3, and PIK3CB is the focus of PPI network. A total of 72 related miRNAs and 130 related TFs were predicted by miRNA and TF targeting network analyses. Ten pairs of gene–drug interaction networks were also predicted; immune infiltration analysis showed that SH3GLB1, ATP6V0E2, PLEKHF1, RALB, KLHL3, and TSPO were closely related to CD8 + T cells and neutrophils. The ROC curve showed that ARGs had good diagnostic value in KD. qRT-PCR showed that WIPI1 and GBA were significantly upregulated.Twenty potential ARGs were identified by bioinformatics analysis, and WIPI1 and GBA may be used as potential drug targets and biomarkers.The online version contains supplementary material available at 10.1186/s41065-023-00278-9. Kawasaki disease (KD) is a vasculitis that primarily affects the coronary arteries . It can Autophagy is an evolutionarily conserved catabolic process that maintains the dynamic balance of the internal environment . Under tIn the present study, we identified ARGs and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, we constructed a protein–protein interaction (PPI) network of differentially expressed genes (DEGs) between the KD and control groups using the STRING database, we predicted ARG-targeted microRNAs (miRNAs) and transcription factors (TFs), and we constructed a gene–drug interaction network. Moreover, we examined immune cell infiltration in KD, and the expression levels of ARGs were analyzed in KD patients and healthy controls. In conclusion, we analyzed the roles of ARGs in KD to understand the pathogenesis of KD.https://www.ncbi.nlm.nih.gov/geo/) [We acquired the KD-related datasets GSE68004  from theov/geo/)  via the ov/geo/) . The Homov/geo/) .Based on the annotation information of GSE68004 and GSE73461 samples, we divided the samples into normal and KD groups. We employed the R package limma to perform differential expression analysis of genes in different groups, setting |log(fold change)|> 1 and Padj < 0.05 as the threshold criteria. Genes with log(fold change) > 1 were upregulated, and genes with log(fold change) <  − 1 were downregulated. The differential expression analysis results were visualized using the R package pheatmap, and heatmaps and volcano maps were plotted using the R package ggplot2. We took the intersection of genes that were differentially expressed in both datasets for subsequent analysis.https://www.genecards.org/) [The GeneCards database (ds.org/)  providesds.org/)  containsP < 0.05 was considered to indicate statistical significance.GO annotation analysis  is a comhttps://cn.string-db.org/) [The STRING database (db.org/)  can be udb.org/) , and scrdb.org/) .To comprehensively and accurately mine the regulatory relationships between mRNAs and miRNAs and between mRNAs and TFs in KD, we predicted the target miRNAs of ARGs in KD according to the miRWalk , miRDB , miRTarBThe Drug–Gene Interaction Database (DGIdb) version 3.0  is an opP < 0.05 to derive the immune cell infiltration matrix. Heatmaps were generated using the R package ggplot2 to show the infiltration of the 22 immune cell types in each sample.CIBERSORT  was usedFrom March 2022 to September 2022, we collected 10 whole blood samples, comprising five KD and five healthy control samples, from the Xianning Central Hospital of Hubei, China. Samples were from patients aged 8 months to 6 years. We collected 3–5 mL whole blood samples in the tubes containing EDTA from all the participants and submitted them to white blood cell (WBC) enrichment. Total RNA was extracted from leukocytes of children with KD and healthy controls, and blood samples from KD children were collected before intravenous immunoglobulin (IVIG) and aspirin treatment. Reverse transcription was performed using the Servicebio® RT First Strand cDNA Synthesis Kit . Quantitative PCR (qPCR) was performed using 2 × SYBR Green qPCR Master Mix (None ROX)  following the manufacturer’s instructions. The thermocycling conditions were as follows: initial activation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 60 °C for 30 s. GAPDH was used as the internal reference for data normalization. Relative expression was calculated by the 2 − ΔΔCt method. Primers are listed in Table Shttps://www.r-cret.org/, version 4.0.2). For the comparison of continuous variables between the two groups, the statistical significance of normally distributed variables was estimated by the independent Student t-test, and the differences between non-normally distributed variables were analyzed by the Mann–Whitney U test . All statistical tests were two-sided. Differences were considered statistically significant at P < 0.05.Receiver operating characteristic (ROC) curve analysis was conducted to predict dichotomous outcome events with continuous variables and to evaluate the degree of good or bad predictor variables. We plotted ROC curves using the R package pROC  and calcA flowchart of the present study is shown in Fig. To identify genes related to autophagy, we searched the GeneCards and MSigDB databases with “autophagy” as the search keyword and downloaded the list of the autophagy-associated genes. We intersected ARGs obtained from the GeneCards and MSigDB databases and obtained 473 ARGs Fig. .We intersected the DEGs in datasets GSE68004 and GSE73461 to obtain 815 DEGs Fig. F. We theWe performed GO enrichment analysis of ARGs in KD. In the biological process category, ARGs were enriched in processes such as autophagy and macro-autophagy. In the cellular component category, ARGs were enriched in components such as the vacuolar membrane and the autophagosome membrane. In the molecular function category, ARGs were enriched in proton-exporting ATPase and GTPase activity. Next, we performed KEGG pathway enrichment analysis of ARGs in KD. ARGs in KD were enriched in autophagy—animal and the collecting duct acid secretion pathway Fig. . The GO We constructed a PPI network behind the ARGs in KD based on the STRING database, which contains a total of 12 nodes and 10 edges Fig. A. We useTo comprehensively and accurately mine the regulatory relationship between mRNA and miRNA and between mRNA and TFs in KD, we predicted the target miRNAs of ARGs in KD using the miRWalk, miRDB, miRTarBase, and starBase databases. miRNAs identified in all four databases were considered to be miRNA targets of ARGs in KD. The target TFs of ARGs in KD were predicted using the hTFtarget and knockTF databases, and TF identified in both databases were considered to be TF targets of ARGs in KD. We constructed miRNA–mRNA and mRNA–TF interaction networks using Cytoscape.From the mRNA–miRNA network, a total of 72 miRNAs were predicted to be targeted by 12 ARGs in KD, and eight genes  were not predicted to target miRNAs Fig. C. From tWe predicted drugs that may interact with ARGs in KD based on the DGIdb database. We obtained 10 gene–drug pairs including three genes  and 10 drugs . We then constructed a gene–drug interaction network using Cytoscape software Fig. E. Among P-values of < 0.05 were considered to indicate statistical significance. In dataset GSE68004, 18 genes  were upregulated and two genes (ATP6V0E2 and HL3) were downregulated in KD patients , GBA (AUC = 0.978), RALB (AUC = 0.975), DRAM1 (AUC = 0.968), GNAI3 (AUC = 0.948), TSPO (AUC = 0.94), WIPI1 (AUC = 0.947), QSOX1 (AUC = 0.946), ATP6V0E2 (AUC = 0.953), ATP6V1C1 (AUC = 0.935), LRRK2 (AUC = 0.908), C9orf72 (AUC = 0.909), PIK3CB (AUC = 0.921), PLEKHF1 (AUC = 0.958), and WDFY3 (AUC = 0.908) had a good diagnostic effect for KD patients  and ATP6V0E2  were negatively correlated with CD8 + T cells, and PLEKHF1  was negatively correlated with neutrophils. The genes SH3GLB1 , RALB , and ATP6V0E2  showed a positive correlation with neutrophils , PLEKHF1 , and KLHL3  showed a positive correlation with CD8 + T cells; and SH3GLB1 , RALB , and TSPO  showed a negative correlation with CD8 + T cells , resting memory CD4 + T cells , and CD8 + T cells and gamma-delta T cells  presented a negative correlation, whereas resting memory CD4 + T cells and CD8 + T cells , activated dendritic cells and resting mast cells , and gamma-delta T cells  presented a positive correlation , M0 macrophages , and neutrophils , whereas M0 macrophages showed a positive correlation with neutrophils  and activated dendritic cells , and M1 macrophages showed a positive correlation with resting dendritic cells  : WIPI1 and GBA were upregulated in KD samples compared with control samples. WDFY3, ATP6V0E2, RALB, KLHL3, C9orf72, LRRK2, GNAI3, and PIK3CB showed no significant difference between KD samples and control samples  from 25 to 4% . Some paAutophagy is a conserved lysosomal degradation process that plays a key role in adaptation to metabolic stress, removal of damaged organelles, pathogen defense, and nutrient reutilization . A recenGO analysis revealed that these ARGs were mainly involved in biological processes such as autophagy and macro-autophagy. KEGG pathway analysis showed that most DEGs were involved in autophagy—animal. Qi et al. found that ginsenoside Rb1 may inhibit CAL inflammation by regulating the AMPK/mTOR/P70S6 autophagy signaling pathway .We then established a PPI network to analyze the interaction of ARGs in KD and screened the top 10 hub genes  using Cytoscape software and the cytoHubba plugin. We also detected potential miRNAs, TFs, and drugs associated with KD, analyzed the expression levels of ARGs in tissue-infiltrating immune cells, and evaluated the diagnostic value of ARGs. These data improve our understanding of the etiology of KD.WD repeat domain, phosphoinositide interacting 1 (WIPI1) is a component of the autophagic machinery and is activated downstream of the ULK1 and PI3 kinases to participate in autophagy. WIPI1 mRNA expression can be used as an indicator of autophagosome formation , 44, WIPMiRNAs are endogenous small, non-coding RNAs. We constructed an mRNA–miRNA network and predicted a total of 72 target miRNAs, some of which have been validated. hsa-miR-19a-3p was suggested by Jone et al. to be a possible biomarker to distinguish KD from other infectious diseases . AnotherIn a rat study, octreotide was shown to exert significant anti-inflammatory effects by reducing the levels of inflammatory cytokines such as TNF-α , and anoWe used the CIBERSORT package to evaluate the types of infiltrated immune cells in KD patients and controls and the correlation with ARG expression. Our analysis showed that SH3GLB1, ATP6V0E2, PLEKHF1, RALB, KLHL3, and TSPO were closely related to CD8 + T cells and neutrophils. A previous study showed that neutrophil counts increased rapidly in the acute phase of KD and decreased significantly in the recovery phase , and a mWe performed ROC curve analysis of autophagy genes in KD to further verify their diagnostic value in KD. In dataset GSE68004, the genes SH3GLB1, GBA, RALB, DRAM1, GNAI3, TSPO, WIPI1, QSOX1, ATP6V0E2, ATP6V1C1, LRRK2, C9orf72, PIK3CB, PIK3CB, and WDFY3 had a good diagnostic effect for KD. In dataset GSE73461, the genes SH3GLB1, GBA, RALB, DRAM1, GNAI3, TSPO, WIPI1, QSOX1, ATP6V0E2, ATP6V1C1, PIK3CB, WDFY3, and KLHL3 had a good diagnostic effect for KD. We finally examined the expression levels of 10 genes by qRT-PCR. The expression levels of WIPI1 and GBA were consistent with our bioinformatics analysis results.There are still some limitations in our study. First, the prevalence of KD varies by race, but KD samples in the GSE dataset are limited to KD samples from Europe and the United States. Second, the small sample size verified via qRT-PCR may reduce the confidence of the study. We will expand the sample size in future studies. Finally, the prognostic power of the identified ARGs remains unclear.A total of 20 ARGs that may be related to KD development were identified by bioinformatics analysis. WIPI1 and GBA may be potential ARGs of KD and should be further verified.Additional file 1: Supplementary Table 1. Primer sequences for quantitative real-time PCR. Additional file 2: Supplementary Table 2. Autophagy-related genes.Additional file 3: Supplementary Table 3. Autophagy-related genes in Kawasaki disease.Additional file 4: Supplementary Table 4. GO and KEGG enrichment analyses.Additional file 5: Supplementary Table 5. Protein–protein interaction network.Additional file 6: Supplementary Table 6. mRNA–miRNA interaction network. Additional file 7: Supplementary Table 7. mRNA–TF interaction network. Additional file 8: Supplementary Table 8. mRNA–drug network.


Microspore embryogenesis is an extraordinarily complicated process, comprehensively regulated by a composite network of physiological and molecular factors, among which hormone is one of the most crucial factors. Auxin is required for stress-induced microspore reprogramming, however, the mechanism of its regulation of microspore embryogenesis is still unclear.− 1 IAA on the buds of Wucai significantly increased the rate of microspore embryogenesis, and moreover accelerated the process of embryogenesis. Physiological and biochemical tests showed that the contents of amino acids, soluble total sugar, soluble protein, and starch were significantly increased after IAA treatment. Furthermore, exogenously spraying 100 mg·L− 1 IAA significantly enhanced IAA, GA4, and GA9 content, increased catalase (CAT) and malondialdehyde (MDA) activity, and reduced abscisic acid (ABA), MDA and soluble protopectin content, H2O2 and O2·− production rate in the bud with the largest population of late-uninucleate-stage microspores. Transcriptome sequencing was performed on buds respectively treated with 100 mg·L− 1 IAA and fresh water. A total of 2004 DEGs were identified, of which 79 were involved in micropores development, embryonic development and cell wall formation and modification, most of which were upregulated. KEGG and GO analysis revealed that 9.52% of DEGs were enriched in plant hormone synthesis and signal transduction pathways, pentose and glucuronic acid exchange pathways, and oxidative phosphorylation pathways.In this study, we found exogenously spraying 100 mg·L2O2 and O2·−. Combined with transcriptome analysis, it was found that most genes related to gibberellin (GA) and Auxin (IAA) synthesis and signal transduction, pectin methylase (PME) and polygalacturonase (PGs) genes and genes related to ATP synthesis and electron transport chain were upregulated, and genes related to ABA synthesis and signal transduction were downregulated. These results indicated that exogenous IAA treatment could change the balance of endogenous hormones, accelerate cell wall degradation, promote ATP synthesis and nutrient accumulation, inhibit ROS accumulation, which ultimately promote microspore embryogenesis.These findings indicated that exogenous IAA altered the contents of endogenous hormone content, total soluble sugar, amino acid, starch, soluble protein, MDA and protopectin, the activities of CAT and peroxidase (POD), and the production rate of HThe online version contains supplementary material available at 10.1186/s12864-023-09483-2. Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of non-heading Chinese cabbage (Brassica campestris L.), which is an important species in the Brassicaceae family [Brassicaceae family crops are cross-pollinated. Microspore embryogenesis is the most effective tool of obtaining doubled haploids (DH) plants that are utilized in plant breeding for accelerating production of new cultivars [Wucai (e family . As an ie family . Breedinultivars .Microspore embryogenesis involve a complex network of factors . There ap < 0.05) hormone in plant development and it is the key regulator of cell division and differentiation [Brassica napus [B. napus, GA3 improved plantlet regeneration, mainly via elongation of the embryo axis and acceleration of its maturation [B. napus embryo at the globular stage of development significantly reduced axis elongation [Abscisic Acid Insensitive 3 (ABI3) and ABI4 transcription factors shown to play a relevant role in embryo formation [Among plant growth regulators, auxin is the most significant  levels have different degrees of influence on microspore development. In previous studies, ROS were involved in the programmed cell death (PCD) of tapetal cells , 21. Thep < 0.05) increased the rate of microspore embryogenesis and moreover accelerated the process of embryogenesis. RNA-seq analysis suggested that plant hormone synthesis and signal transduction pathways, pentose and glucuronic acid exchange pathways, and oxidative phosphorylation pathways were involved in the regulation of microspores embryogenesis. Combined with analysis of changes in endogenous hormones and physiological indicators and transcriptome analysis, it was found that exogenous IAA treatment could change the balance of endogenous hormones, accelerate cell wall degradation, promote ATP synthesis and nutrient accumulation, inhibit ROS accumulation, which ultimately promote microspore embryogenesis.In this study, the optimal concentration of exogenous IAA was identified in Wucai, which significantly (p < 0.05) increased treated with IAA  and Route B . Route A can be further subdivided according to the second division and subsequent situations. Studies had shown that there were two pathways, A and B, for embryonic development in isolated microspore cultures of non-heading Chinese cabbage, and pathway A was more common .In this study, the microspore mother cells underwent meiosis to form a tetrad Fig. , and fou− 1 IAA treatment.Compared to the control, the IAA treated microspores had a dispersed arrangement of four microspores during the tetrad stage Fig. b. At thep < 0.05) decreased after exogenous IAA treatment, while the content of other phytohormones did not show obvious (p < 0.05) changes /ABA, (auxin + GAs + CKs)/ABA, GAs/ABA, and auxin/ABA were all higher in the treatment group than in the control group  greater than or equal to 1, and the p-value less than 0.05. A total of 2004 DEGs were identified, comprising 1002 upregulated genes and 1002 downregulated genes. In addition, genes that were not compared to the reference genome were considered new, and we identified 345 new genes, 52 of which could be not annotated to any database.To better understand the expression patterns of differentially expressed genes (DEGs), DESeq was used for differential expression analysis , 31. DEGDetailed GO term annotations of the DEGs were categorized according to biological processes, molecular functions, and cellular components. The GO categories for the set of DEGs Fig.  revealedwww.kegg.jp/kegg/kegg1.html ) enrichment analysis showed that 1334 differential genes were enriched in 121 metabolic pathways , the transcription factors PIF3 and PIF4 were activated to mediate the gibberellin signal response. GA4 , non-bioactive GAGA4 Fig. E and GA9. 9 Fig. F contentCYP71AB was downregulated, and DAO was upregulated. There were four genes encoding aldehyde dehydrogenase (ALDH), of which one was upregulated and three were downregulated. During auxin signal transduction (ko04075), more than 60% of DEGs were upregulated. At the same time, we found that the content of IAA was significantly (p < 0.05) higher than that in the control group , tryptophan is catalyzed by enzymes to synthesize indole 3 acetamide (IAM) and indole 3 acetaldehyde (IAAld), prerequisite substances of IAA. IAA1d is synthesized into indoleacetic acid through the action of aldehyde dehydrogenase. In our study, LUT5), β-carotene isomerase (DWARF27), and abscisic β-glucosyltransferase (AOG) were all downregulated, while abscisic acid 8’-hydroxylase (CYP707A) was upregulated. Leading to a decrease in ABA accumulation in vivo. Both the abscisic acid receptor (PYL) and protein phosphatase (PP2C) were downregulated during signal transduction (ko04075). This was consistent with our detection of a significant reduction (p < 0.05) in ABA content , β-cyclohydroxylase  decreased H2O2  in plants supplies the needs for plant growth and breeding. We plotted a model of oxidative phosphorylatipn metabolism Fig. A. A tota2O2 Fig. C, MDA  after treated with IAA.To further prove that the development of microspores was related to the energy supply, the energy substances and nutrient substances were measured. Compared with the control group, the total soluble sugar Fig. A, amino PME) and polygalacturonase (PGs), key enzymes in pectin metabolism, encoding polygalacturonases (GSVIVT) and UDP-sugar pyrophosphorylase (USP)  lower than that of the control  was significantly enriched. The regulatory pattern of related differential genes in the metabolic pathway was shown in Fig. SP) Fig. C. The perol Fig. B.IAA26, D27, DAO, GA2OX2, CYP71A13, ABAH3, C7352, GH33, CED3), pentose and glucuronic acid exchange pathways , oxidative phosphorylation metabolic pathways . The gene expression patterns obtained from RT-qPCR and RNA-Seq data showed similar trends, which confirmed the accuracy of the RNA-Seq results obtained in this study.16 DEGs were selected from the important metabolic pathways for RT-qPCR to verify the reliability of RNA-Seq Fig. . These DBrassicaceae species [ALDH gene has the function of overcoming this phenomenon [CYP71A13 is to catalyze the conversion of IAOx to IAN [BGLU) [CYP71A13 was downregulated, and BGLU was upregulated; one gene encoding ALDH was upregulated. During plant hormone signal transduction, SAUR, and AUX/IAA, which are annotated as auxin-responsive proteins, are upregulated with high signal transduction levels. Related research shows that AUX prevents auxin leakage by transporting extracellular auxin back to the cytoplasm [Hormones are an important factor affecting the development of microspore embryos. Auxin biosynthesis, transport, and response severely affect embryo development  IAA can  species –39. Premenomenon . The prix to IAN . The regN [BGLU) . In thisytoplasm . This ma− 1 IAA, the contents of endogenous GAs, GA4and GA9 were significantly increased. In the GA biosynthesis process, the expression of GA20OX and GA2OX regulates the level of GA with biological activity in vivo. The molecular mechanism of GA on microspore embryogenesis needs to be further studied.Studies have shown that exogenous IAA applications can improve endogenous GA biosynthesis . The proLUT5 results in a decrease in beta-carotene-derived lutein [DWARF27 is a protein that converts trans-carotene to 9-cis-carotene [NCED) plays a key role in the development of ABA synthesis in plants [CYP707A) is a key enzyme for ABA catabolism [DWARF27, LUT5, NCED, and abscisic beta-glucosyltransferase (AOG) were downregulated, while CYP707A was upregulated. Both abscisic acid receptors (PYR/PYL) and protein phosphatase 2 C (PP2C) were downregulated, i.e., so ABA synthesis and transcription levels were low. This was consistent with significant decrease in ABA content.ABA accumulation inhibits the occurrence of microspore embryos, and low concentration of ABA promotes the occurrence of microspore embryos . The absd lutein . DWARF27carotene . 9-Cis-en plants . Abscisitabolism . ABA see− 1 IAA was statistically analyzed  play important roles in mitochondrial electron transport. NADH dehydrogenase complex 1 was an enzyme that catalyzes the transfer of electrons from NADH to CoQ at the inner mitochondrial membrane, and was the “entry enzyme” for mitochondrial oxidative phosphorylation . Succinaynthesis . We specPME) demethylates pectin to form pectic acid and methanol, which alters the pH value of the cell wall and relax it [PME genes are involved in pollen development in Arabidopsis and cabbage[PGs) uses polygalacturonic acid as the substrate. It acts on alpha-1, 4-D- galactosides bond to hydrolyze the pectic acid on the polygalacturonic acid main chain into galacturonic acid, galacturonic acid, the main component of pectin in the plant cell wall, is degraded into a single polygalactose residue which exists in tapetum, sporophyte, and pollen [PME and PGs were upregulated, and in addition, the pectin content was significantly lower than that in the control group. Therefore, we speculated that upregulation of key genes involved in pectinase accelerated pectin degradation, resulting in a decrease in pectin content, degradation of the cell wall of pollen mother cells, and accelerated release of tetrad microspores. Pectate lyase (PLY) is present in anthers and pollen and can loosen cell walls [Cell proliferation, expansion, and differentiation are the essential cytological basis for plant growth and development. There is a close link between this series of cytological events and cell wall metabolism. As a significant component of the primary cell wall of higher plants, the metabolism of pectin modification, synthesis, and degradation was involved in numerous aspects of plant growth and development by affecting the mobility and malleability of the cell wall . Pectin relax it . Severald cabbage. Polygald pollen , 67. In ll walls . USP catll walls –71. USP ll walls .PLY and USP were upregulated genes, and we speculate that upregulation of PLY may lead to partial autolysis of cell wall and loosening of cell wall. The upregulation of USP may have accelerated cell growth, which may further lead to an increased incidence of isolated microspore embryogenesis.In this study, 75% of WUS, GSTU12, PEX3, PRK3, BGL13, BFRUCT3, PME5, CYP707A3, AHA8, AHA9, PPA3, and PPA6 were highly expressed in the IAA treated group. WUS plays an important role in the embryonic development of Arabidopsis thaliana [GST is related to the embryonic development of sorghum [Exploring the genes involved in the initiation of microspore embryos will help to better understand the molecular mechanism of the molecular mechanism of microspore embryogenesis. In this study, we further screened 139 DEGs from genes related to metabolic pathways and development, 97 of which were upregulated. thaliana , and a s sorghum . We spec− 1 IAA was the optimal hormone concentration for inducing isolated microspore embryogenesis by counting the embryo yield. Cytological observation and the statistics of initial time of embryogenesis indicated that IAA treatment accelerated the development of microspores. Treatment with exogenous IAA caused changes in the content and dynamic balance of endogenous plant hormones. We have focused on the transcript and pathway, particularly affecting embryonic development. Transcriptome analysis, it was found that most genes related to GA and Auxin (IAA) synthesis and signal transduction, PMEs and PGs genes, and genes related to ATP synthesis and electron transport chain were upregulated, and genes related to ABA synthesis and signal transduction were downregulated. Physiological and biochemical indexes showed that exogenous IAA altered the contents of soluble total sugars, amino acids, starch, soluble protein, malondialdehyde and pectin, CAT and POD activities, and the production rates of H2O2 and O2·−. These results indicated that exogenous IAA treatment could change the balance of endogenous hormones, accelerate cell wall degradation, promote ATP synthesis and nutrient accumulation, and inhibit ROS accumulation, which ultimately promotes microspore embryogenesis.In this study, we first determined that 100 mg·LLine Huiwu (HW) was screened by Chenggang Wang, and all seed materials were kept in the Vegetable Breeding Laboratory of the College of Horticulture, Anhui Agricultural University . No specific permits were required for the described field studies. The location is not privately-owned or protected in any way, and the field studies did not involve endangered or protected species.The Wucai varieties HW grew under 25℃ and light intensity was 20,000 lx conditions in the plastic greenhouse of the Wanjiang Vegetable Industry and Technology Research Institute  in the spring.− 1, 50 mg·L− 1, 75 mg·L− 1, 100 mg·L− 1, 125 mg·L− 1, 150 mg·L− 1 IAA and collected after 24 h. Bud pre-treatment was performed according to method of Zhang et al. [5 liquid medium using sterile glass rods and filtering the suspension through a 30 μm nylon mesh. Centrifugation was performed three times , and the microspores were resuspended in isolation medium. The centrifuged microspores were collected and resuspended in NLN-13 medium, with 13% sucrose and buffered at pH 5.8.The microspore density was adjusted to 1 × 105 microspores per milliliter using a hemocytometer  [At the full bloom stage (25-75% of the whole plant flowers open period), the buds at the mononuclear stage were treated with 0 mg·LGermany) . The mic− 1 IAA and the control group. Representative microspores cultured for 0,3,5,10, and 15 days were selected from the IAA treatment and the control group respectively, with three replicates. The microspores in 3 petri dishes were randomly selected together with the medium and inhaled into a 20 mL centrifuge tube, centrifuged at 800 rpm for 4 min, and the supernatant was discarded. After collection, the microspores were sucked onto the slide, covered with the cover slide, cut into pieces, and observed under an inverted microscope.Cytological examination of the microspores for the first two weeks of culture after treatment with 100 mg·L− 1 IAA or the fresh water respectively by using LC-MS/MS method, including Auxin, CKs, GA, JA and ABA. 0.3 g fresh buds were collected, immediately frozen in liquid nitrogen, ground into powder , and stored at -80 °C. 50 mg of plant sample was weighed into a 2 mL plastic microtube and frozen in liquid nitrogen, dissolved in 1 mL methanol/water/formic acid . 10 µL internal standard mixed solution (100 ng/mL) was added into the extract as internal standards (IS) for the quantication. The mixture was vortexed for 10 min, then centrifugation for 5 min , the supernatant was transferred to clean plastic microtubes, followed by evaporation to dryness and dissolved in 100 µL 80% methanol (V/V), and filtered through a 0.22 μm membrane filter for further LC-MS/MS analysis [Six hormones were selected and determined in flower buds treated with 100 mg. Lanalysis –79.The chromatographic column was a Waters ACQUITY UPLC HSS T3 C18, 1.8 μm, 2.1 mm × 100 mm. The mobile phase (phase A) was ultrapure water with 0.04% acetic acid, and the organic phase (phase B) was acetonitrile with 0.04% acetic acid. The elution gradient was set as follows: 0 min, A: B = 95:5 (V: V); 1 min, A: B = 95:5 (V: V); 8 min, 5:95 (V: V); 9 min, 5:95 (V: V); 9.1 min, 95:5 (V: V); and 12 min, 95:5 (V: V). The flow rate was 0.35 mL/min, the column temperature was 40 ◦C, and the injection volume was 2 µL.Mass detection using Q-Trap 6500+ . The mass spectrometry conditions were as follows: electrospray ionization (ESI) temperature, 550℃; mass spectrum voltage, 5500 V; curtain gas (CUR), 35 psi. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® 6500 + LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in both positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites –82.− 1 IAA and the control group were extracted using RNAprep Pure Plant Plus kit  according to the manufacturer’s instructions. RNA degradation and contamination was monitored on 1% agarose, RNA purity was checked using the NanoPhotometer® spectrophotometer . RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina®  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. After the completion of library construction, the Qubit2.0 Fluorometer was used for initial quantification, followed by Agilent 2100 bioanalyzer for insert size of the library. After the insert size met the expectation, qRT-PCR accurately quantified the effective concentration of the library (the effective concentration of the library was higher than 2nM) to ensure the quality of the library. The clustering of the index-coded samples was performed on a Bot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and 150 bp paired-end. Use fastp v 0.19.3 to filter the original data, mainly to remove reads with adapters, all subsequent analyses are based on clean reads. Use HISAT v2.1.0 to compare clean reads with Brassica rapa v 3.0 reference genome. Use String Tie v1.3.4d for new gene (genes not compared to reference genome) prediction. Use featureCounts v1.6.2 /StringTie v1.3.4d to calculate the gene alignment and FPKM. DESeq2 v1.22.1 /edgeR v3.24.3 was used to analyze the differential expression between the two groups, and the P-value was corrected using the Benjamini & Hochberg method. The corrected P-value and |log2foldchange| are used as the threshold for significant difference expression. The enrichment analysis was performed based on the hypergeometric test. For KEGG, the hypergeometric distribution test is performed with the unit of pathway; for GO, it was performed based on the GO term.Total RNA from buds of treatment with 100 mg·L− 1 of IAA between 8: 00 a.m. and 9: 00 a.m. at the full bloom stage (25-75% flowering period of the whole plant). The control treatment was sprayed with fresh water under the same conditions. After 24 h, buds with the largest population of late-uninucleate-stage microspores stage were selected for determination of soluble sugar, soluble protein, starch, ROS related enzymes and Oxidative damage index. The experiment was repeated three times. The soluble sugar content was determined by an anthrone colorimetric method, soluble protein content was determined by Coomassie brilliant blue G-250 method [The buds were treated with 100 mg. L0 method , and sta0 method .A centrifuge tube was used to collect 50 mg dry flower bud sample, which was mixed with 4 mL of 80% ethanol. The sample was treated with a fresh water bath at 80 ℃ for 30 min, cooled and centrifuged at 5000×g for 3 min. The supernatant was collected, and the residue was washed with 80% ethanol three times. After each washing, the supernatants were extracted and combined. Then 10 mg of activated carbon was added, decolorized at 80 ℃ for 30 min, diluted to 10 mL, and then filtered to obtain the extract.20 µL of the above extractive solution was combined with 480 µL of fresh water and 2.5 mL of anthrone, mixed, reacted in a 90℃ fresh water bath for 15 min, taken out, cooled rapidly, and develop for 10 min before being measured at the wavelength of 620 nm.10 mL of 52% perchloric acid was added into the above residue, the residue was centrifuged at 4000×g for 3 min, and the supernatant was collected. Then 10 mg of activated carbon was added, and the residue was treated with a fresh water bath at 80℃ for 30 min. After decolorization, filtration was performed, 5 mL of anthrone was added into 2 mL of filtrate, and the filtrate was shaken up immediately. Then the filtrate was treated with a fresh water bath at 80℃ for 10 min, cooled, developed in the dark for 10 min, and then the absorbance value was measured at a wavelength of 620 nm.− 1 pre-cooled phosphate buffer solution (pH 7.8) was added and ground on an ice bath to homogenate, then transferred to a centrifuge tube for centrifugation at 4℃ and 12,000×g for 20 min. To 20 uL of the extract, 80 uL of fresh water was added, followed by 2.9 mL of Coomassie brilliant blue solution. After reacting for 2 min, the absorbance was measured at 595 nm.After washing, 0.2 g of fresh buds were placed in a pre-cooled mortar, 1.6 mL of 5 mmol. L2·−), hydrogen peroxide (H2O2) content and catalase (CAT), peroxidase (POD) activities, Protopectin content were determined using Solarbio reagent kits . Each sample (fresh buds) about 50–200 mg (fresh weight) thoroughly ground for the determination of the above indicators.Amino acid (AA), MDA content, superoxide anion  was reverse transcribed into cDNA using a PrimeScript™ RT kit . RT-qPCR was performed using SYBR® Premix Ex Taq™ II kit . PCR amplification was performed in a Bio-Rad CFX96 instrument according to the manufacturer’s instructions. Three biological replicates were performed for each sample, and three technical replicates were performed for each gene. The data were standardized with the expression level of BnaActin as the control. The relative expression levels were calculated using the 2 −ΔΔCt method. Primer Software Version v6.0  was used to design primers, and primer sequences were listed in additional file 8: Table RNA was isolated from the flower buds with single nucleus sidelined treated with 100 mg. Lt tests with a significance level of 0.05. Graphs were drawn using GraphPad Prism software , Origin 2018 64 Bit, and Adobe Illustrator CC 2019.All data are expressed as the mean ± standard deviation, using at least three biological replicates. Data were statistically analyzed using spass.26  software, and means were compared using the Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8