Neothada, including N. cancellata, N. hades and N. major, collected from the rhizosphere of mosses in Khuzestan, southwestern Iran, are redescribed and illustrated. Neothada hades and N. major are new records from Iran. Neothada hades has 14 longitudinal incisures excluding the lateral field, body length of 586 (505–674) µm, stylet 10.5 (10.0–10.8) µm in length bearing distinct basal knobs, and an elongated-conical tail 70.4 (65–74) µm long with a finely to bluntly rounded terminus. N. major possesses 18–20 longitudinal incisures excluding the lateral field, body length of 657 (600–728) µm, stylet 10.9 (10.3–11.7) µm long with basal swellings but not distinct knobs, and an elongated-conical tail 78.2 (70–83) µm long ending to a finely to bluntly rounded terminus. Molecular phylogenetic studies of the two species (N. hades and N. major) with 664 bp of D2-D3 expansion segments of 28 S rDNA revealed that they form a clade with N. cancellata.Three species of the genus  Neothada to accommodate Thada cancellataT. tatraNeothada and Thada in the subfamily Thadinae Neothada currently contains six valid species namely: N. tatra  N. cancellata  Pers. and Prosopis cineraria (L.) Druce, in Dezful and Bostan, both cities in Khuzestan province, southwestern Iran. Nematodes were extracted with the tray method  each of forward and reverse primers, 2 μl of DNA template and 11 μl deionised water. This mixture was placed into a Hybaid Express thermal cycler . The thermal cycling profile was denaturation at 95°C for 4 minutes, then 33 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 90 seconds. A final extension was performed at 72°C for 10 minutes. The quality of PCR was checked by electrophoresis of 4 μl of the PCR reaction in 1% agarose gel containing ethidium bromide. Products were visualized and photographed under UV light. The length and concentration of each PCR product was measured by comparison with a low DNA mass ladder . The PCR product was purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer . The newly obtained sequences were submitted to GenBank database under accession numbers MN970001 and MN970002 for the D2-D3 expansion fragments of 28 S sequences.Nematode DNA was extracted from single individuals and DNA extracts were stored at −20°C until use as PCR template. Protocols for DNA extraction were followed as described by Aphelenchus avenae6 generations. The tree was visualized and saved with FigTree 1.4.3  of the total stylet length, 7–8 annules from anterior end; knobs conspicuous, rounded, 1.7–2.2 µm wide. Dorsal pharyngeal gland opening 2.6–4.0 µm from stylet base. Corpus cylindroid with slightly swollen median bulb lacking valve; isthmus as wide as procorpus, nerve ring at mid-isthmus and located 56–73 µm from anterior end. Pharyngeal basal bulb short, pyriform, 7.5–9.0 µm wide, 19–24 µm in length. Pharyngo-intestinal valve hemispherical. Excretory pore slightly sclerotized, at middle of basal bulb, 38–45 annules from anterior end. Hemizonid one annule anterior to the excretory pore, 85–104 µm from anterior end. Deirids adjacent to the level of excretory pore, 87–107 µm from anterior end. Vulva a transverse slit, not protruding, without lateral flaps. Vagina width 6.7–8.0 µm, 37–46% of vulva body diameter. Post-vulval uterine sac length 9.0–12.8 µm or 57–63% of vulval body diameter. Spermatheca long, variable in shape, near-rectangular, 7.5– to 9.0 µm × 26 to 33 µm. Ovary outstretched, oocytes arranged in a single row. Rectum curved to slightly sigmoid, length half of anal body diameter. Tail elongate-conoid, tail tip finely to bluntly rounded, 27–33 annules on ventral side of the tail.Not found.In all, 10 females are deposited in the nematode collection of the Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran.Soil around of mosses in Dezful, Khuzestan Province, southwestern Iran, by Manouchehr Hosseinvand at February 2017 .Neothada hades can be distinguished from all other known species of the genus by possession of distinct stylet knobs. The morphology and morphometrics of the Iranian population are coincident with the original species description of Heyns & Van den Berg (1996), except for the number and width of body annules , tail length (65–74 vs 55–66 µm) and absence of males (vs presence).Neothada major Maqbool & Shahina, 1989  (Jand) in Bostan, Khuzestan Province, southwestern Iran, by Manouchehr Hosseinvand, March 2019 .Soil around N. major, except for minor differences in stylet length (10.3–11.7 vs 12–14.4), c ratio (7.5–8.7 vs 9.0–10.2) and absence of males (vs presence).The morphology and morphometrics of the Iranian population agree well with the type population of Neothada cancellata , c ratio (7.5–8.6 vs 8.8–11.0), V (70–71.8 vs 72.8–75.6) and absence of males (vs presence).The morphology and morphometrics of the Iranian population agree completely with the data given by Cynodon dactylon (L.) Pers. and mosses in Dezful, Khuzestan Province, southwestern Iran, by Manouchehr Hosseinvand at February 2017 .Soil around roots of N. cancellata (KP730046), and 80–82% similarity with Basiria spp. using BlastN search in NCBI. The partial D2-D3 of 28 S rDNA gene sequences alignment contained 38 taxa including Aphelenchus avenae (KP527123) as outgroup taxon and was 664 bp in length after removing ambiguously aligned regions. The 50% majority rule consensus phylogenetic tree generated from the partial D2-D3 region of 28 S rDNA alignment by Bayesian inference (BI) analysis under GTR + I + G model is presented in Two new D2-D3 28 S rDNA gene sequences were obtained in the present study (MN970001 and MN970002). These sequences showed a 96–97% similarity values to N. hades is closely related to N. cancellata (PP = 100) and both species are related in the clade with N. major (PP = 100). N. hades differs from N. cancellata by 16 bp (2.1%) and from N. major by 11 bp (1.6%), and N. major differs from N. cancellata by 20 bp (1.9%).Neothada is highly supported as a valid genus ; Boleodorus and two sequences of Neopsilenchus magnidens  formed a basal clade with them. Discopersicus iranicus (KM502982) and two sequences of Neopsilenchus , other likely members of the subfamily Boleodorinae, are placed in other clades in the tree. According to Thada forms a clade with FilenchusThada along with Neothada in the subfamily Boleodorinae. We believe that in the subfamily Boleodorinae, the distance of dorsal esophageal gland orifice (DGO) from the stylet base and development of median bulb and stylet knobs are also important diagnostic characters as much as the shape of amphidial apertures. In the genus Neothada, the number of longitudinal incisures, degree of stylet knobs development and tail tip shape are important diagnostic characters for identification of the known species.In our 28 S rDNA phylogeny, id genus , similarid genus , and as


Clostridium butyricum is a spore-forming probiotic which can promote the enhancement of beneficial bacteria and maintain intestinal microecological balance. However, it is difficult to improve the production level of C. butyricum by conventional fermentation process. In this study, a co-fermentation process of C. butyricum DL-1 and Bacillus coagulans ZC2-1 was established to improve the viable counts and spore yield of C. butyricum, and the formula of coculture medium was optimized by flask fermentation. The results showed that the optimum medium composition is 10 g/L bran, 15 g/L corn steep powder, 15 g/L peptone, 1 g/L K2HPO4 and 0.5 g/L MnSO4.Cultured stationarily in the optimal medium for 36 h, the number of viable bacteria of C. butyricum DL-1 reached 1.5 × 108 cfu/mL, Which was 375 times higher than that incubated in the initial medium. The sporulation rate reach 92.6%. The results revealed an economical and effective medium composition for the coculture of C. butyricum and B. coagulans, which achieved a 64.6% cost reduction. The co-fermentation process established in this study provides a new fermentation mode for the industrial production of other absolute anerobic bacteria.The online version contains supplementary material available at 10.1186/s13568-022-01354-5. A novel co-culture system was established.C. butyricum and B. coagulans was optimized.Medium composition for the coculture of The optimal medium cost is lower than what was reported.The online version contains supplementary material available at 10.1186/s13568-022-01354-5. Bacillus  and 1492R (5′-GGTTACCTTGTTACGAC T T-3′). The PCR protocol consisted of the following steps: 5 min at 94 °C for the first denaturation step, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min, and ended with a final extension step at 72 °C for 7 min. The PCR reaction mixture (20 μL) consisted of an appropriate amount of DNA template (10–100 ng), 1.0 μL of Taq DNA polymerase , 0.5 μL of 10.0 μM each primer, and 8.5 μL of ddHMultiple sequence alignments were performed with ClustalW in MEGA 6, and phylogenetic trees were constructed from the evolutionary distance data calculated from Kimura’s two-parameter model using the neighbor-joining method by MEGA 6. Bootstrap analyses were performed based on 1000 random resampling. Reference sequences were retrieved from GenBank with the accession numbers indicated in the trees.B. coagulans inoculum was cultured in proliferation medium at 37 °C and 180 rpm for 16 h. C. butyricum DL-1 inoculum was cultured at 37 °C stationarily for 16 h, with the shake flasks sealed with 8 layers of gauze and 2 layers of kraft paper. The co-fermentation condition was as follows: loading liquid ratio was 40%, inoculation ratio of C. butyricumDL-1 and B. coagulans strains was 6% and 4%, respectively. The shake flask was sealed with 8 layers of gauze and 2 layers of kraft paper were cultured stationarily at 37 °C. After a 24 h culture, the viable counts and spore concentration were analyzed.C. butyricum, 100 µL of samples were spread on agar plates. The colonies formed after incubation at 37 °C for 16 h were counted and statistically analyzed. Viable counts were expressed as colony-forming units per milliliter (cfu/mL). Spore concentration was measured by the same method as that of viable counts except that the cell suspension was heated at 80 °C for 10 min in advance. Spore yield was calculated as the percentage of spore count to total viable cells of C. butyrium DL-1. Three replicates were set for each dilution.After sequential tenfold dilution of cell suspensions of C. butyricum DL-1 proliferation medium inoculated at a 4% inoculum size was cultured stationarily at 37 °C, with a 60% filling volume. OD600 and pH of the culture broth were assayed every 4 h. Growth process of B. coagulans ZC2-1 was monitored by the same protocol as that of C. butyricumDL-1, except that the inoculated proliferation medium was cultured at 180 rpm, with a filling volume of 20%.Specific C. butyricumDL-1. The factors were studied successively, and the optimization results were used in subsequent experiment step. Bran, corn starch, soluble starch, sucrose, lactose, maltose and glucose were used as alternative carbon source. Corn steep powder, soybean meal powder, peptone, beef extract, fishmeal, tryptone, yeast extract and yeast were used as alternative nitrogen source. NaCl, KCl, K2HPO4, CaCO3, MnSO4, MgSO4, and Sodium acetate trihydrate [C2H3NaO2·(H2O)] were used as candidate inorganic salts. Viable counts and spore yield of C. butyricum DL-1 were analyzed at regular intervals with the methods described above. Three parallel experiments were conducted for each experimental group.Single-factor tests were used to study the effect of carbon source, nitrogen source, and inorganic salts on the viable counts and spore yield of 9(33) orthogonal experiment was designed to optimize the concentration of carbon source and nitrogen source, and a L9(32) orthogonal experiment was designed to study the effect of inorganic salts concentration on the viable counts and spore yield of C. butyricum DL-1. The orthogonal experiment was designed by SPSS 20.0 software.Based on the results of single-factor tests, a LPolyphasic taxonomic analyses of the strains were conducted based on their phenotypic characteristics and phylogenetic analysis Fig. . Strain B. coagulans ZC2-1, ZA-1, ZB-1, ZC-9  were deposited in GenBank with the accession numbers MW195020, MW504830, MW504831, MW504832, respectively. Among them, strain B. coagulans ZC2-1 has been stored in China General Microbiological Culture Collection Center with the preservation numbers of CGMCC No. 22951.16S rRNA sequences of the four B. coagulans strain (MT604689.1). The 16S rRNA sequence of ZA-1 and ZB-1 revealed 100% identity with the B. coagulans strain MT611810.1 and MT611733.1, respectively. And the 16S rRNA sequence of ZC-9 has a 99.93% identity with the B. coagulans strain MT626077.1. ZC2-1, ZA-1, ZB-1, ZC-9 was located in the same B. coagulans clade , there is a 20.75 time improvement  orthogonal table  orthogonal experiment  and MnSO4 (B) content as factors.K2HPO4 is the most important influence factor for the viable bacteria and spore rate of C. butyricum DL-1. The optimum inorganic salt combination was 1 g/L K2HPO4 and 0.5 g/L MnSO4. Under this condition, the viable counts and spore rate of C. butyricum DL-1 reached 1.3 × 108 cfu/mL and 92.3%, respectively. The number of viable bacteria increased 325 times.According to the range analysis results ) and results analysis of composite; Fig. S1 Growth characteristics and pH change trend of strains C. butyricum DL-1; Fig. S2 Growth characteristics and pH change trend of strains B. coagulans ZC2-1; Fig. S3 The effect of nitrogen source sorts on the viable counts and spore yield of C. butyricum.


Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions. Despite the apparent differences between the nervous and immune systems, they share an intriguing similarity: both employ a system of cytokines and neurotrophic factors enabling them to transmit information from one system to the other . This neA prominent role in neuroimmune crosstalk has been proposed for neurotrophin NGF and p75NTR, a universal neurotrophin receptor with a single transmembrane domain , 8. OrigH2-prone asthma model and alleviating disease progression in a TH1-prone diabetes model  , 12. DenA standard method to study protein-protein interactions is immunoprecipitation (IP). Its principle is to capture a target protein (and interacting proteins) by use of a specific antibody coupled to a stationary matrix [for review see ]. ExtensThe receptor p75NTR is an excellent example of a transmembrane protein that poses a challenge to endogenous IP experiments with subsequent mass spectrometric detection. The molecular mass of p75NTR amounts to approx. 42 kDa based on the amino acid composition. The protein runs between 65 to 80 kDa in denaturing SDS-PAGE as it is subject to extensive post-translational modification with intra- and intermolecular disulfide-linkages, O- and N-glycosylation –28. In cThis comprehensive protocol collection details the workflow to successfully immunoprecipitate p75NTR at endogenous, low abundance expression levels. We validated the specificity of three monoclonal p75NTR antibodies  and IP conditions in human melanoma cell line A375, a model cell line for these conditions , 48. We Our proposed workflow consisted of the following steps:confirmation of p75NTR mRNA transcription by quantitative RT-PCR and protein expression by flow cytometry and immunofluorescence in both model and target cell linesantibody validation in cell line A375 by IP, western blotting and mass spectrometry,application of this optimized protocol to immunoprecipitate p75NTR and potentially interacting proteins in pDC-like cell line PMDC05.Mouse hybridoma cell line 2003G64  producing p75NTR antibody ME20.4, American Type Culture Collection, Manassas, VA, USAHuman melanoma cell line A375, a kind gift from Friedegund Meier and Dana Westphal, Clinic of Dermatology, University Clinic Carl Gustav Carus, Dresden, GermanyHuman leukemic plasmacytoid dendritic cell line PMDC05, provided by Miwako Narita , Kana Sakamoto and Kengo Takeuchi  Cell culture reagents and chemicals are detailed in Details about the use of antibodies in immunofluorescence and flow cytometry are listed in Goat anti-Rabbit IgG-Alexa Fluor 555 (A-21429), chicken anti-mouse IgG-Alexa-Fluor 647 (A-21463), goat anti-Mouse IgG-Alexa Fluor 555 (A-21424), Chicken anti-Rabbit IgG-Alexa Fluor 647 (A-21443) were purchased from Thermo Fisher Scientific. HRP-linked Donkey anti-Rabbit IgG (NA9340) was obtained from GE Healthcare.Elution Buffer: 0.1 M glycine in distilled water (pH 2.4 adjusted with HCl).Lysis Buffer: 130 mM NaCl, 50 mM HEPES (pH 7.4), 1% (v/v) Triton X-100 in distilled water. Supplemented with 1 tablet protease inhibitor cocktail (Roche cOmplete Ultra Mini EDTA-free) per 10 mL.4% (w/v) PFA solution: dissolve 4% (w/v) PFA in HBSS, incubate at 80°C until solution becomes transparent, cool on ice. Store at 4°C and use for a month.2 0.14 g L-1, MgCl2 · 6 H2O 0.1 g L-1, MgSO4 · 7 H2O 0.1 g L-1, KCl 0.4 g L-1, KH2PO4 0.06 g L-1, NaCl 8 g L-1, Na2HPO4 · 7 H2O 0.09 g L-1, D-glucose 1 g L-1 in ddH2O.HBSS: CaCl-1, KH2PO4 0.2 g L-1, NaCl 8 g L-1, Na2HPO4 1.15 g L-1 in ddH2O.PBS: KCl 0.2 g LPBS++: 0.5% (w/v) BSA and 2 mM EDTA in PBS.-1 DAPI in PBS.PBS-DAPI: 0.1 µg mLPermeabilization Buffer: 0.1% (v/v) Triton X-100 and 1% (w/v) BSA in PBS.-1 penicillin-streptomycin.RPMI complete: RPMI 1640 supplemented with 10% (v/v) FCS, 2 mM GlutaMAX and 100 units mL2O.SDS Sample Buffer (2×): 5% (w/v) SDS, 25% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol, 0.25 mM Tris (pH 6.8) in ddHTBS-T: 0.05% (v/v) Tween 20 in TBS .TBS-T-M: 5% (w/v) nonfat dry milk powder in TBS-T.Washing Buffer: 130 mM NaCl, 50 mM HEPES .10× Tris buffered saline .Both cell lines A375 and PMDC05 must be handled at biosafety level 1: wear personal protective equipment  at all times.Adhere to additional biosafety regulations of your institution, especially when transfected or infectious cell lines are used.4 cells cm-2 in pre-warmed RPMI complete (0.2 mL cm-2) in an incubator .Cultivate at a density of 2-5 × 10Passaging -2), swivel cell culture vessel and discard supernatant.a. remove cell culture supernatant, gently add PBS (0.1 mL cm-2) and incubate at 37°C for 5 min, check cell detachment with microscope.b. add pre-warmed 21 µM trypsin solution (0.03 mL cmc. add 3 volumes of RPMI complete.d. transfer cell suspension to a centrifuge tube and centrifuge at 300 × g for 8 min at RT.e. resuspend cells in pre-warmed RPMI complete and count cells (e.g. in a Neubauer chamber).f. adjust cells to desired concentration.6 cells cm-2 in pre-warmed RPMI complete (0.2 mL cm-2) in an incubator .Cultivate at a density of 0.25-0.5 × 10Passaging -2) to loosen slightly attached cells and centrifuge at 300 × g for 8 min at RT.a. transfer cell suspension to a centrifuge tube, rinse surface of the culture vessels with PBS (0.05 mL cmb. resuspend cells in pre-warmed RPMI complete and count cells (e.g. in a Neubauer chamber).c. Adjust cells to desired concentration.Viable cell concentration can be determined by trypan blue exclusion.PMDC05 are sensitive, slowly growing cells and tend to form clusters. At the beginning, they may struggle to regenerate from the thawing process. Once stable growth has been observed subcultivation intervals may be increased to 7 days.Transfer cells to centrifuge tubes and perform the following steps at 4°C.Wash cells twice by centrifugation at 300 × g for 8 min and resuspension in ice-cold 1x PBS.For RNA preparation:6 cells to a 1.5 mL reaction tube.a. Transfer 5 × 10b. Centrifuge at 500 × g for 8 min.c. Carefully remove supernatant as completely as possible.d. Store dry pellets at -80°C.4. For whole cell protein lysates:7 cells to a 1.5 mL reaction tube.a. Transfer 2-10 × 108 cells mL-1) e.g. by trituration.b. Lyse cells in lysis buffer  according to the manufacturer’s instructions. Content and purity of RNA preparations was assessed on a microvolume spectrometer  by calculating 260 nm/280 nm absorbance ratios. Complementary DNA (cDNA) was synthesized using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific) with oligo-dT primers according to the manufacturer’s instructions. Quantitative PCR was performed on a QuantStudio 5 Real-Time-PCR-System (Thermo Fisher Scientific) using the TaqMan system (Thermo Fisher Scientific) according to the manufacturer’s instructions. Assay probes were purchased from Thermo Fisher Scientific designed to cover exons 1-5 of human p75NTR  at a concentration of 2 × 10Transfer 50 µL of cell suspension to a 96-well plate cavity.Incubate for 10 min.Add 200 µL PBS++, centrifuge for 8 min at 300 × g and discard the supernatant.Resuspend in 55 µL PBS++ containing the appropriate antibody concentration see Table 1.Incubate for 30 min in the dark.Add 200 µL PBS++, centrifuge for 8 min at 300 × g and discard the supernatant.If using fluorescent dye labeled primary antibodies, proceed to step 13.If using primary-secondary staining:a. Wash cells again with 200 µL PBS++.b. Resuspend in 55 µL PBS++ containing the appropriate secondary antibody see Table 1.c. Incubate for 30 min in the dark.d. Add 200 µL PBS++, centrifuge for 8 min at 300 × g and discard the supernatant.13. Resuspend in 200 µL PBS++.-1.14. Transfer to a FACS tube and add an equal amount of PBS-DAPI resulting in a working DAPI concentration of 0.05 µg µL15. Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences). Data were analyzed using FlowJo .An important step when handling immune cells for flow cytometry consists in blocking receptors that may bind Fc parts of immunoglobulins. FcR blocking reagents should always be included to prevent unspecific antibody binding.Remember to include unstained and isotype controls.When staining with multiple dyes, compensation for interfering fluorescence signals is recommended.-1. Although general recommendations on the use of DAPI in flow cytometry exist, working concentrations should be validated in the specific experimental condition.Typical DAPI working concentrations range from 0.05-2 µg µLBased on their fluorescence intensity, different cell subsets can be sorted . Th 1. Coat sterile precision coverslips (Carl Roth) with poly-L-lysine (Merck)-2).a. Add 0.01% (v/v) poly-L-lysine solution to coverslips a. PMDC05 coverslips were centrifuged in 24-well plates at 300 × g for 5 min at each washing step.4. Fix cells in 4% (w/v) PFA solution for 15 min at 37°C.5. Remove 4% (w/v) PFA solution and wash cells three times for 5 min with HBSS on a shaker.-1) in HBSS for 10 min at 37°C.6.Counterstain the membrane with WGA  BSA in PBS and PMDC05 cells for 60 min in 1% (w/v) BSA in PBS containing human FCR Block (1:80), and 10% goat/mouse serum (depending on the dye-labeled primary and secondary antibody).10. Stain cells with the primary antibodies in 1% (w/v) BSA in PBS.11. Incubate for 2 hours (A375) at RT or overnight (PMDC05) in a wet chamber in the dark at 4°C.12. Wash A375 cells three times in HBSS for 5 min on a rocking platform at medium speed.a. Wash PMDC05 cells three times by centrifugation in 24-well plates at 300 × g for 5 min.13. Stain cells with the secondary antibody in 1% (w/v) BSA in PBS.14. Incubate for 60 min at RT in a wet chamber in the dark.15. Wash A375 cells three times in HBSS for 5 min on a rocking platform at medium speed.a. Wash PMDC05 cells three times by centrifugation in 24-well plates at 300 × g for 5 min.-1 in HBSS for 15 min at 37°C.16. Counterstain DNA with 1 µg Hoechst 33342 mL17. Wash A375 cells twice in HBSS for 5 min and once in distilled water on a rocking platform at medium speed.a. Wash PMDC05 cells twice in HBSS and once in distilled water by centrifugation in 24-well plates at 300 × g for 5 min.18. Remove coverslips carefully and mount them with VECTASHIELD Antifade Mounting Medium on glass slides.Microscopy was performed according to Weidemuth et al. . BrieflyPMDC05 cells basically grow in suspension and therefore adhere to coated coverslips weakly. To avoid losing cells during the staining procedure shortened washing and centrifugation is mandatory. That might affect cell integrity and thus antibody localization. The risk of losing adherent A375 cells after fixation is minimal.Permeabilization and fixation chemicals might influence epitope accessibility and therefore antibody binding. Different protocols for permeabilization and fixation [e.g. ] may be 1. Perform all steps on ice/at 4°C (unless indicated otherwise).2. Resuspend 1.5 mg magnetic protein G Dynabeads in PBS in 1.5 mL reaction tube.3. Wash beads in following procedurea. Put on a static magnet , collect beads by incubation for 5 min.b. Carefully aspirate supernatant and discard without disturbing collected beads.c. Remove bead containing reaction tube from static magnet.d. Resuspend beads by gently pipetting up and down in 500 µL PBS and repeat washing steps from a to c twice.4. For antibodies MLR2 and D4B3:a. Prepare 200 µL PBS containing 2 µg D4B3 and 10 µg MLR2, respectively.b. resuspend beads by gently pipetting up and down in antibody coupling solution.5. For antibody ME20.4:a. resuspend beads gently in 10 mL serum free hybridoma culture supernatant.6. Prepare coupling solutions containing the same concentration of isotype control antibody.a. resuspend beads by gently pipetting up and down in antibody coupling solution.7. Incubate magnetic beads for 2 h at room temperature under constant agitation to prevent the beads from settling down.8. Put on a static magnet, collect beads by incubation for 5 min.9. Carefully aspirate supernatant and discard.10. Remove bead containing reaction tube from static magnet.7 cells) and adjust volume to 500 µL by adding lysis buffer, if necessary.11. Add 200 µL protein lysates .22. For a second elution (fraction E2), repeat steps 17-21.23. Resuspend beads in 100 µL SDS sample buffer.24. Heat to 95°C and incubate for 5 min under constant agitation.25. Put on a static magnet, collect beads by incubation for 5 min and save eluate (fraction col).Careful resuspension of magnetic beads ensures the bead integrity. But especially after coupling antibodies, harsh treatment, e.g. vortexing, must be avoided to preserve stable binding and capture.Addition of detergent to all washing solutions and coupling buffers prevents clumping of beads and may reduce unspecific binding if downstream applications permit this.For mass spectrometry, any detergent must be washed out before analysis. This is ensured by washing and elution in detergent-free buffer.If captured protein amounts are low, consider washing in detergent-free buffers. The use of crosslinking agents, e.g. dithiobis succinimidyl propionate, is compatible with many downstream applications.Heating protein samples is a critical step when eluting in SDS sample buffer. Hydrophobic membrane proteins may precipitate when heated above 70°C. Avoid long incubation times at high temperatures as peptide bonds may hydrolyze.Add 10 µL 2× SDS sample buffer to 10 µL sample solution.Incubate at 95°C for 10 min under constant agitation.Load samples and protein standard on gels and run electrophoresis for 35 min keeping voltage constantly at 200 V.Transfer gel proteins to Immobilon-P transfer membrane in a wet chamber for 90 min keeping voltage constantly at 30 V.Perform all following steps under constant agitation (e.g. on a rocking platform).Wash membrane three times for 5 min in 20 mL TBS-T.block membrane in 20 mL TBS-T-M for 15 min at room temperature.Incubate with D4B3 antibody buffer (1:1000 in 4 mL TBS-T-M) overnight at 4°C.Wash membrane three times for 5 min in 20 mL TBS-T.Incubate in secondary antibody solution  for 1 hour at room temperature.Wash membrane three times for 5 min in 20 mL TBS-T.-2) to PVDF membrane.Add Lumi-Light PLUS western blotting substrate .Addition of dyes to SDS sample buffers may ease loading protein samples onto gels.Some proteins may be heat-sensitive (see above). Lower temperatures if necessary.Larger proteins transfer from gel to membrane more slowly than smaller ones. Protein transfer may be assessed by gel staining with Coomassie or silver-based dyes. Transfer times may be extended if protein transfer is insufficient. Membranes with small pore sizes (0.22 µm) may be sensible when smaller proteins are lost during transfer.If capture and detection antibody are derived from the same species HRP-linked protein A/G may be used instead of a secondary antibody. This approach minimizes unspecific detection of denatured capture antibodies Figure 3A positive control is recommended when sampling unknown antibodies in western blot. These positive controls can be manufactured from cell lines/tissues with a well detectable expression of the protein in question. In our case, an example is cell line A875. If such a cell line or tissue is not available an alternative strategy is the generation of a cell line with a transgenic expression (see For information on the instrumentation and software applied see Resuspend beads in 100-200 µL suitable buffer for proteolytic digestion (e.g. 20-50 mM Tris-HCl or HEPES pH 7.5).-1) and incubate overnight (12-18h) at 37°C in a shaker to avoid settling of the beads.Add 2 µL sequencing grade Trypsin (100 ng µL-1) and incubate again overnight at 37°C in a shaker.Add additional 2 µL sequencing grade Trypsin (100 ng µL-1 for rLys-C from Promega) and incubate again overnight at 37°C in a shaker.Add 2 µL sequencing grade Lys-C (e.g. 50 ng µLSpin-down beads and collect supernatant.Acidify supernatant with 2% TFA (pH<2) and desalt on C18 ultramicro column (e.g. Nest Group or Harvard Scientific Ultramicro Spin-Columns C18) according to the manufacturer’s instructions .Dry the eluate of the desalting column in a speed vac and store the dry peptide mixture at -20°C until LC-MS/MS analysis.Dissolve the peptide mixture in an appropriate volume formic acid  and transfer the solution into an HPLC vial for LC-MSMS analysis.-1 and linear gradients of water, 0.1% formic acid (A) and acetonitrile, 0.1% formic acid achieve the high sensitivity recommended for this kind of analysis. Commonly the mass spectrometer is operated in data dependent acquisition mode selecting the most intense peptide ions for fragmentation automatically.Proteomic LC-MS/MS is commonly performed with a nanoflow UPLC system hyphenated directly to a mass spectrometer. Low flow rates of 200-300 nL minThe peptide fragmentation data is extracted from the raw MS data files and submitted to a data interpretation program that identifies the peptides and the proteins present in the sample.Quantification of proteins can be performed to gain insight into quantitative changes between samples rather than judgment by presence or absence of proteins to differentiate true interactors from experimental background.To support data interpretation the enrichment of protein identifications with additional information, e.g. GO molecular function or localization, may prove helpful and suitable commercial and academic programs are available.The protease quality is essential for efficiency and specificity of the digestion. Sequencing grade proteases are recommended.Trypsin is generally available as stabilized  enzyme that reduces auto-digestion of the enzyme.-1) may vary depending on the manufacturer.Lys-C is available as native and recombinant enzyme, and the specific activity  and acetonitrile, 0.1% formic acid. Different companies offer this kind of LC systems and specific parameters for equilibration time, gradient steepness and handling may apply.Proteomic LC-MS/MS is commonly performed with nanoflow UPLC system operated at flow rates of 200-300 nL minDifferent mass spectrometers are suitable for mass spectrometric analysis of complex peptide mixtures and differ in resolution, acquisition speed and peptide fragmentation methodology. In general, a typical approach for the bottom-up proteomic LC-MS/MS analysis is the fragmentation of the most intense multiply charged  peptide ions (TopN) in data-dependent acquisition mode.Peptide and protein identification from mass spectrometric raw data requires extraction of fragment spectra, matching of experimental spectra with theoretical spectra or spectral libraries and a variety of academic and commercial software are available , 60, 62.gapdh for quantification of RNA expression. All analyzed exons are expressed in cell lines A375 and PMDC05 with an average expression compared to gapdh of 0.26 and 0.29%, respectively, indicating a low expression in both cell lines.A prerequisite for the analysis of protein-protein interactions is the detection of p75NTR expression on RNA and protein level. The p75NTR gene has six exons, as annotated in the reference transcript NM_002507.3 . We used). Isotype controls and single channel images as well as images of permeabilized cells are provided in the supplementary information , ME20.4 (mouse IgG1) and MLR2 (mouse IgG2a). Cells were prepared and labeled for flow cytometry according to protocol. In a strict gating strategy with two singlet gates and a DAPI-based dead cell exclusion gate , first elution fractions  from the same immunoprecipitations were analyzed by mass spectrometry. Given the amino acid sequence of p75NTR, its extensive post-translational modifications and our protein digestion techniques, the number of reliably detectable peptides is limited. Theoretical prediction resulted in four detectable peptides. Expert opinion excluded one based on an extremely low detection probability due to its length. All three p75NTR specific peptide sequences were detected in acid elution fractions from immunoprecipitations with antibodies D4B3, ME20.4 and MLR2 Table 3 In summary, we have shown that all three antibodies detect p75NTR specifically and reliably in low abundance model cell line A375. Due to the very specific band pattern of ME20.4, we chose the combination of ME20.4 for capture and D4B3 for detection antibody in step 3.7 cells were prepared. IPs were performed using the ME20.4-D4B3 antibody combination and the resulting western blots are shown in 6 cells , a grant from Deutsche Forschungsgemeinschaft , the MeDDrive program of the Faculty of Medicine Carl Gustav Carus at Technische Universitaet Dresden (to A-CF), the Saxon State Ministry for Science and the Arts (to ST), and the Dr. Robert Pfleger Foundation Bamberg (to SB and ST). The Facility ‘Molecular Analysis - Mass Spectrometry’ at the CMCB was generously supported by grants of the European Regional Development Fund (ERDF/EFRE) (Contract #100232736), the German Federal Ministry of Education and Research  and the German Research Foundation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Poecile carolinensis Audubon is a relatively small songbird belonging to the tit and chickadee family Paridae. Feces from three P. carolinensis from Polk County, Arkansas, USA, and a single P. carolinensis in McCurtain County, Oklahoma, USA, were collected and examined for coccidia; the latter bird was found to be passing a new species of Isospora. Oöcysts of Isospora oklahomaensis n. sp. are subspheroidal to ovoidal with a smooth to slightly-pitted bi-layered wall, measure (L × W) 32.1 × 28.3 μm, and have a length/width (L/W) ratio of 1.1; a micropyle and oöcyst residuum were absent but a bilobed and refractile polar granule is present. Sporocysts are ellipsoidal and measure 18.4 × 11.8 μm, L/W 1.6; a prominent Stieda body is present as well as a distinct sub-Stieda body. The sporocyst residuum is composed of an irregular mass of granules lying between and dispersed among the sporozoites. This is the first coccidian described from the Carolina chickadee and, most importantly, only the second described from a member of the Paridae, worldwide.The Carolina chickadee,  Poecile carolinensis (Audubon) is a relatively small passerine belonging to the tit and chickadee family Paridae. It possesses a black cap and bib with white cheeks and formerly belonged to the genus Parus (L.) but was transferred to Poecile Kaup by P. carolinensis atricapilloides (Lunk) is found statewide except for the Panhandle. It seldom decends to the ground and prefers valleys and foothills in open deciduous woodland, forest clearings and edge, swamps, thickets, second-growth woodland, parks, brushy areas,and suburban areas. Carolina chickadees eat insects, especially moths and caterpillars, insect eggs, spiders, fruits, and seeds , Polk County, Arkansas, USA . In addition, fresh feces were collected from a single  P. carolinensis taken from a bird box in April 2021 in Hochatown, McCurtain County, Oklahoma, USA ; after defecation, all birds were released unharmed. Fecal samples were placed in individual vials containing 2.5% (w/v) aqueous potassium dichromate (K2Cr2O7). After flotation in Sheather’s sugar solution (specific gravity = 1.30), they were examined for coccidia using an Olympus BX53 light microscope . One bird was found to be passing unsporulated and partially sporulated oöcysts and the sample was placed in a Petri dish containing a small layer of K2Cr2O7 for 48‒72 h to allow sporulation. It was further examined using Olympus© cellSens 1.14 digital software (https://www.olympus-lifescience.com/en/software/cellsens/) and all morphological measurements are reported in micrometers (μm) with the means followed by the ranges in parentheses. Photographs were taken using Nomarski interference-contrast optics at ×1,000 magnification. Oöcysts were ca. 30 days old when measured and photographed.Fresh feces were collected from 3 Descriptions of oöcysts and sporocysts follow the standard guidelines of A single Carolina chickadee was found to be passing a coccidian that we describe herein as new.Eimeriidae Minchin, 1903IsosporaSchneider, 1881Isospora oklahomaensis n. sp.Isospora rara Poecile carolinensis  (Aves: Passeriformes: Paridae), adult female collected 15 April 2021.Hochatown, McCurtain County, Oklahoma, USA .Photosyntypes of sporulated oöcysts are deposited as HWML 216542.1 of 4 (25%) overall; 1/1 McCurtain County, Oklahoma, USA; 0/3 (0%) Polk County, Arkansas, USA.All oöcysts were fully sporulated within 48‒72 hrs.Unknown; oöcysts were passed in feces.Isospora oklahomaensis n. sp. is urn:lsid:zoobank.org:act:BD2187AA-9ED5–4DE3–8ABF-F0B4470814BB.To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature , detailsokla and humma, meaning “honored people”. The new name is formed as an adjective, feminine.The specific name is derived from the US state locality for the coccidian, Oklahoma, the 46th state to enter the union on November 16, 1907. The state’s name is derived from the Choctaw words Description , 2A–DOöcyst (n = 15) subspheroidal to ovoidal; (32.1 × 28.3) 29–36 × 27–30, length/width (L/W) ratio 1.1–1.2. Wall smooth to slightly-pitted, thick, bilayered, tan to yellow, c.2.0 (1.3–2.1), outer c. 1.2 (0.8‒1.4), inner 0.7 (0.5‒0.8). Micropyle and oöcyst residuum absent but single bilobed and refractile polar granule (3 × 2.7) present.Sporocysts (n = 15) 2, ovoidal 18.4 × 11.8 (16–20 × 9–13); L/W ratio 1.6 (1.4–1.7); wall smooth, thin and uni-layered, light brown, c.0.5 thick. Prominent Stieda and distinct sub-Stieda bodies present, para-Stieda body absent; sporocyst residuum spheroidal granules of various sizes dispersed between sporozoites. Sporozoites (4), vermiform 12.7 × 3.6; L/W ratio 3.3; subspheroidal anterior and ellipsoidal posterior refractile bodies, nucleus in centre of sporozoites.Poecile Kaup currently recognized in the tit family Paridae Vigors  from India has been described worldwide from birds of this family (Isospora sp. (see There are seven species of the genus e Vigors . Surpriss family  (Table 1 sp. see .Isospora lacazei (Isospora sp. (Cyanistes caeruleus (L.) and great tit, Parus major (L.) from the Czech Republic  from Great Britain  from passeriform birds are problematic as they were classified as  lacazei , some otpora sp. . In addiRepublic  and oöcy Britain , Table 2I. parusae, there are three major differences: (1) oöcysts of the new species are considerably larger (32.1 × 28.3 vs. 24.2 × 20.8), (2) I. parusae possesses a micropyle that the new species clearly does not, and (3) the hosts are from different, widely-separated continents. In addition, when compared to coccidians reported from New World passerine birds of the Superfamily Sylvioidea  (I. oklahomaensis is a new species.When the new species is compared to ropidae) , oöcystsropidae) . TherefoParus palustris (L.), six P. montanus, and a single coal tit, Parus ater (L.) from the Czech Republic but none were passing coccidians at the time they were examined. It is obvious that novel coccidian parasites have been rarely described as novel species among members of the Paridae. This is an enigma because this family is a large and widespread group of 64 species of small passerine birds which mainly occur in Europe, Asia, North America, and Africa (P. carolinensis as well as the first described from a parid bird in the Western Hemisphere.d Africa . Therefo


Furthermore, cytochrome P450 (CYP) activity was investigated using the cocktail probe method. The results revealed that Na-DHA exhibited an inductive effect on CYP1A2, 2D1/2, and 3A1/2 activities by changing the main pharmacokinetic parameters of probe drugs in male rats. However, no significant change in CYP2E1 activity was found. There were sex differences in the metabolism and coagulation in rats exposed to Na-DHA. The lower metabolism and higher blood Na-DHA concentration in females may be the reasons for higher coagulation sensitivity in female rats.Sodium dehydroacetate (Na-DHA), a fungicide used in food, feed, cosmetics, and medicine, has been found to cause coagulation aberration accompanied by the inhibition of vitamin K epoxide reductase (VKOR) in the liver in rats. VKOR complex 1 (VKORC1) and VKORC1 like-1 (VKORC1L1) are two homologous VKOR proteins. Little information is available on the effect of Na-DHA on VKORC1L1 in the liver or VKORC1/VKORC1L1 in extrahepatic tissue and sex differences in Na-DHA metabolism. In the present study, after administration of 200 mg/kg Na-DHA by gavage, significant inhibition of VKORC1 or VKORC1L1 expression in tissues, as well as prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT), were observed. The PT/APTT in the Na-DHA-exposed males were 1.27- to 1.48-fold/1.17- to 1.37-fold, while the corresponding values in the Na-DHA-exposed females were 1.36- to 2.02-fold/1.20- to 1.70-fold. Serum or tissue Na-DHA concentrations were significantly higher in females than in males. The pharmacokinetic parameters (t The formation or function of VK-dependent clotting factors is known to be directly affected by the VK cycle . The redf VKORC1 . Vkorc1 d testis . There ad testis . The mRN in rats .Alicyclobacillus acidoterrestris-related spoilage in the fruit juice/beverage industry (The main structure of sodium dehydroacetate (Na-DHA), 2H-pyran-2-one, is similar to that of warfarin. Na-DHA is widely used as a preservative in food because of its antimicrobial effect. It is commonly used in baked food and pickled vegetables. Furthermore, it is known to well control industry . Na-DHA industry . It has industry . The accHemorrhage in multiple organs and prolongation of blood coagulation time have been reported in Sprague-Dawley (SD) rats after repeated oral administration of Na-DHA . In our Na-DHA  was purchased from Sigma-Aldrich . Phenacetin (Cat. No. 100095), omeprazole , chlorzoxazone , and dapsone  were obtained from the Institute of Standard Substances, Chinese Academy of Sciences . Wistar rats, from the Comparative Medicine Center of Yangzhou University, were housed under the following controlled conditions: temperature of 22°C, relative humidity of 40–60%, and a 12 h light-dark cycle. All experimental protocols were reviewed by the Committee for the Ethics of Animal Experiments of Yangzhou University  and were performed per the Regulations for the Administration of Affairs Concerning Experimental Animals, China, and the EU Directive 2010/63/EU for animal experiments.Na-DHA (200 mg/kg) was administered once daily by gavage for 9∼11 days and normal saline was administered to the control group. The rats were anesthetized by intraperitoneal injection of 2% thiopental sodium. Blood, liver, lung, kidney, and testis or ovary samples were collected for determining Na-DHA concentration by using high-performance liquid chromatography (HPLC) and for analyzing VKORC1/VKORC1L1 expression. Six male and six female rats were used at every sampling point.Blood samples were obtained from the posterior orbital venous plexus of the rats and mixed with trisodium citrate immediately at a volume ratio of 9:1. Plasma was collected by centrifugation and used to determine PT and APTT with a PT assay kit or APTT (ellagic acid type) assay kit  by using a blood coagulation analyzer  according to the manufacturers’ instructions.HPLC analysis for Na-DHA concentration measurement was based on the method described by The HPLC conditions were as follows: a Waters X bridge C18 column  at 30°C, a mixture  of methanol and 0.02% ammonium acetate  was used as the mobile phase at 1.0 ml/min; the injection volume was 20 μl and the detection wavelength was 293 nm.R2 = 0.9999 at 0.2–10.0 mg/L). The recovery of Na-DHA was 81.94–88.21% and 88.20–93.61% from blank tissue samples spiked with 0.2 mg/kg, and the intra- and inter-day variations were lower than 5.0 and 6.0%, respectively. The limit of detection was 0.2 mg/kg, and the limit of quantification was 0.8 mg/kg.A standard curve for Na-DHA was y = 200.4x + 8.3425, where y is the Na-DHA chromatographic peak area and x is the Na-DHA concentration in mg/L  in conjunction with oligo-dT primers and hexanucleotides. Real-time PCR was performed using 2 μl of cDNA and SYBR®Premix ExTaq™ II  using the ABI Prism 7,700 Sequence detection system and the relative quantification method. All assays were performed in duplicate.Isolation of mRNA from excised tissues was performed using the MiniBEST Universal RNA Extraction Kit  according to the manufacturer’s recommendations. The purity and quantity of the RNA were determined by spectrophotometry using NanoDrop ND-1000 . ODVkorc1, Vkorc1l1, and β-actin genes in Gene Bank, the following primers were designed: Vkorc1 F-5ʹGGACGTTGGGCCTCTATCCT3ʹ, R-5ʹCAACATCAGGCCCGCATTGA3ʹ; Vkorc1l1 F-5ʹCTCCAGATGGGGTCGAGGATT3ʹ, R-5ʹGCTGGCTGTCATGCCAAGTAA3ʹ; and β-actin F-5ʹAGATCAAGATCATTGCTCCTCCTG3ʹ, R-5ʹCGCAGCTCAGTAACAGTCCG3ʹ. All primers were synthesized by Sangon Biotech Co. Ltd. . The qRT-PCR cycle program was as follows: 1 cycle of 30 s at 95°C, 40 cycles of 3 s at 95°C, 30 s at 60°C, and 5 s at 72°C, with 1 cycle of final extension for 1 min at 72°C.Based on the sequence of rat Tissue samples were washed three times with pre-cooled phosphate-buffered saline (PBS); 0.1 g of tissue and 3 ml of pre-cooled PBS were fully ground in a glass homogenizer and transferred to a 10 ml centrifuge tube. Centrifugation was performed at 4,000 rpm for 3 min at 4°C. The deposit was washed with 3 ml of pre-cooled PBS and then centrifuged at 4,000 rpm for 10 min at 4°C. The sediments were lysed in 1 ml of lysis buffer  for 30 min on ice. After centrifugation at 4,000 rpm for 10 min at 4°C, the supernatant was collected in a 1.5 ml EP tube and then centrifuged at 12,000 rpm for 10 min at 4°C. The protein concentration of the final supernatant was determined and adjusted, and a constant protein concentration (30 μg/lane) was used. The proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane  by using a Bio-Rad Transblot apparatus . After being blocked with 5% skim milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, the membranes were then incubated overnight at 4°C with primary rabbit polyclonal VKORC1 antibody , rabbit polyclonal VKORC1L1 antibody , or mice monoclonal β-actin antibody. Thereafter, the samples were incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system . The grey levels of the bands were assayed using Gel-Pro Analyzer four software .The blood samples were collected from the animals’ jugular vein at 0, 0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 9, 11, and 24 h after Na-DHA (200 mg/kg) was singly administered by gavage. The plasma Na-DHA concentration was detected using the HPLC method described above, and the pharmacokinetic parameters were obtained using the WinNonlin software version 5.3.Six male and female rats were randomly divided into the Na-DHA group to which 200 mg/kg Na-DHA was administered once daily by gavage for 5 days and the control group, which was administered normal saline. All rats were administered the probe drugs by gavage at 10 ml/kg at 24 h after last administration, with the concentration of dapsone, phenacetin, and chlorzoxazone being 2 mg/ml and that of omeprazole being 4 mg/ml. Blood samples were collected at designated time intervals  from the jugular vein; the sample at 0 h was collected directly before the administration of the probe substances. The plasma samples were stored at −20°C until analysis.The concentrations of the four probe substrates for cytochrome P450 (CYP) isoforms were simultaneously analyzed using an Agilent 1200 HPLC system. The HPLC system included the Agilent C18 column . Acetonitrile was used as mobile phase A and 0.02 mM phosphate buffer (pH 5.8) was used as phase B. The gradient procedure was as follows: 0∼11.0 min, 30∼45% A; 11.0∼12.0 min, 45∼30% A; 12.0∼13.0 min, 30% A; temperature of the column box of 30°C; detection wavelength 0∼7.2 min, 293 nm; 7.2∼8.5 min, 245 nm; 8.5∼10.0 min, 302 nm; 10.0∼13.0 min, 280 nm; injection volume of 20 μl; and flow rate of 1.0 ml/min.2. The residue was dissolved in 200 μl of the initial mobile phase through vortexing for 3 min and screening with a 0.22 μm filter for HPLC analysis.The plasma samples for probe drug determination were treated as follows. Acetonitrile (300 μl) was added to 100 μl of the plasma samples and vortexed for 3 min; centrifugation was performed at 14,000 rpm for 15 min, and the clear supernatant was collected. Duplicate extraction of the sublayer was performed by repeat addition of 300 μl of acetonitrile, after which the supernatant was combined and evaporated to dryness by using NThe standard curves of the four probe drugs were determined using the HPLC method and read at 0.15∼5.0 mg/ml for dapsone, phenacetin, and omeprazole and at 0.18∼6.0 mg/ml for chlorzoxazone. The recovery of control plasma spiked with 0.15, 0.5, and 2.5 mg/ml phenacetin, omeprazole, and dapsone, and 0.18, 0.75, and 3.0 mg/ml chlorzoxazone was performed. Furthermore, the intra-day precision and lower limits of quantification (LLQ) of the method used for the four probes were determined. The plasma concentrations of the four probe drugs were determined based on the corresponding curves, and the pharmacokinetic parameters of the drugs were analyzed using WinNonlin5.3 software.p < 0.05 was considered statistically significant.All data were statistically analyzed by GraphPad Prism8 software and expressed as mean ± SM values. One-way analysis of variance (ANOVA) followed by the Turkey post hoc test or Kruskal-Wallis test was used to assess the significance of the differences; the Pearson method was used for correlation analysis. p < 0.05). The values of PT ratios were 1.27∼1.48 in males and 1.36∼2.02 in females, the APTT ratio in male rats was 1.17∼1.37 and that in female rats was 1.20∼1.70, compared to the corresponding values in the control group during 7–9 days treatment. PT or APTT were obviously longer in females than in males, especially during days 7–9 of Na-DHA treatment.The PT and APTT ratios in the rats treated with 200 mg/kg Na-DHA compared to that in the control group are shown in p < 0.05). The Na-DHA concentration ratios in female/male rats were 1.64- to 3.21-fold in the liver, 3.09- to 4.00-fold in the lung, and 1.14- to 1.45-fold in serum during days 3–9 of the treatment. The concentration of Na-DHA in serum achieved a steady-state after 1 week.The Na-DHA concentrations in the serum, liver, lung, and testis of the rats treated by 200 mg/kg are shown in Vkorc1/Vkorc1l1 gene expression in the liver, lung, testis, or ovary of rats treated with 200 mg/kg Na-DHA for different administration times are shown in Vkorc1/Vkorc1l1 genes in different tissues of male or female rats were all significantly reduced at different times of Na-DHA administration (p < 0.05 or p < 0.01) in comparison with the control. Over 3–9 days of administration, the Vkorc1 levels relative to those in the control were 0.69- to 0.93-fold and 0.57- to 0.87-fold in the liver and lung of males, and 0.75- to 0.96-fold in the testis; 0.33- to 0.93-fold and 0.27-to 0.95-fold in the liver and lung of females, and 0.55- to 0.83-fold in the ovary, respectively. The Vkorc1l1 levels were 0.75- to 0.95-fold and 0.48- to 0.89-fold in the liver and lung of males, and 0.72- to 0.92-fold in the testis, respectively. The corresponding levels in the liver, lung, and ovary of females were 0.62- to 0.91-fold, 0.46- to 0.94-fold, and 0.68- to 0.93-fold, respectively. At the same time point of Na-DHA administration, the Vkorc1 levels in the liver or lung of female rats were lower than the corresponding levels in male tissues, and the differences were significant at 7 and 9 days of administration (p < 0.05). The Vkorc1l1 mRNA levels in the liver of female were significantly lower than those in the male liver at 7 or 9 days of administration (p < 0.05).The results of qRT-PCR detection of The results of western blot analysis for VKORC1/VKORC1L1 expression in the liver, lung, testis or ovary of rats treated by 200 mg/kg Na-DHA for different administration times are shown in 1/2), Cmax, area under the concentration time curve (AUC0∼24 h), and mean residence time (MRT0∼24 h) in female rats were significantly higher than those in male rats (p < 0.05), while the apparent volume of distribution (Vz) was significantly lower than that in the controls (p < 0.05), implying differences in pharmacokinetic parameters between male and female rats exposed to Na-DHA and higher inhibition in female rats than in males in terms of metabolism and elimination.The main pharmacokinetic parameters after a single administration of 200 mg/kg Na-DHA are shown in R2 = 0.9999 for dapsone; y = 81.26x + 0.4075, R2 = 0.999 for phenacetin; y = 40.117x–0.8312, R2 = 0.9993 for omeprazole; and y = 26.836x + 0.1015, R2 = 0.9999 for chlorzoxazone. The recovery, intra-day precision, and LLQ of the method are shown in The standard curves of the four probe drugs were y = 116.15x + 2.4972, max and AUC0→t in male rats were significantly lower than those in females, decreasing by approximately half, respectively, while the Vz in male rats was higher than that in female rats (p < 0.05), showing that Na-DHA in multiple dose could significantly speed up the metabolism of phenacetin in males, and Na-DHA had the potential to induce male rat CYP1A2 activity in in vivo. The corresponding parameters of omeprazole in males, namely, t1/2, tmax, Cmax, and AUC0→t, were significantly lower than those in females (p < 0.05), with reductions of about 60% in t1/2 and tmax, 50% in Cmax, and 72% in AUC0→t, indicating that Na-DHA induced significant CYP2D1/2 activity in males. With regard to the effect of Na-DHA on rat CYP2E1 isoform, the pharmacokinetic parameters of chlorzoxazone showed no significant difference between male and female rats, except that the tmax of chlorzoxazone in males was significantly lower than that in females, implying no effect or a slight effect of Na-DHA on the rat CYP2E1 isoform. The pharmacokinetic analysis of dapsone showed that the tmax, Cmax, and AUC0→t in males significantly decreased by approximately 67, 54, and 52%, respectively, while the Vz increased about 2.85-fold compared to that in females (p < 0.05), indicating that Na-DHA accelerates the metabolism of dapsone in male rats and significantly induces CYP3A1/2 activity in male rats. The pharmacokinetic data of the four probe substances in rats validated sex-related differences in the effects of Na-DHA on the CYP activity of rats in in vivo.A cocktail of multiple probe drugs was used to evaluate the effect of Na-DHA on the activity of CYP enzymes in rats. The results of pharmacokinetic analysis of multiple probe drugs in male and female rats are presented in In early study, coagulation abnormality was reported with the inhibition of VKOR activity by Na-DHA . Our furp-values for PT and APTT being 0.033 and 0.046 at 7 days and 0.039 and 0.0116 at 9 days of administration in ANOVA analysis. The Na-DHA concentrations in the serum or tissues of females were also higher than those in males . However, the VKORC1 inhibition rate in the female lung was higher than that in the male lung whereas the inhibition degree in the liver or sex organs in males or females was similar. On the inhibition of VKORC1L1, the inhibition degree in the same tissue in males or females showed greater differences, being slightly more in the liver and sex organ and slightly less in the lung of males in comparison with the corresponding values in females. In male or female rats, the inhibition of VKORC1 was larger than that of VKORC1L1 in the liver and lung and less than that of VKORC1L1 in the testis or ovary  in the male liver was much far less than the translational inhibition (the relative protein levels being less than 0.5-fold during administration of Na-DHA for 7∼11 days). The inhibition of Na-DHA on Vkorc1 and Vkorc1l1 mRNA level in testis and ovary were all far less than their protein level  were more than those in males , in p < 0.05), in PT and APTT are usually used to evaluate coagulation function. Na-DHA was found significantly lengthen the PT and APTT values in male and females, and appeared to exert an anticoagulation effect. The findings of a correlation analysis of PT/APTT with the Na-DHA levels in blood and liver are presented in This study was set to explain why female rats were more sensitive than male rats to Na-DHA. The findings demonstrate that the effects of Na-DHA on the activity of male CYP enzymes or the higher CYP activities in males than in females resulted in slower metabolism and higher blood concentrations of Na-DHA, longer PT/APTT values, and finally more serious coagulation disorders in females than in male rats.We observed sex differences in the metabolism and coagulation function of rats exposed to Na-DHA. Female rats showed more sensitivity to Na-DHA than males may be because of such sex differences in metabolism, effects on CYP enzyme activity, inhibition of VKORC1/VKORC1L1 expression, and prolongation of PT/APTT values.


Streptomyces rochei strain showed a higher hydrolytic zone (24 mm) than the other isolates. Growth under optimized culture conditions using Plackett-Burman's experimental design led to a 1.7, 9.8, 7.7, and 3.12-fold increase for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement. When applying the Box-Behnken design on S. rochei using the most significant parameters , there was a 12.22-fold increase in the specific activity measurement 7.37 U/mg. The α-amylase was partially purified, and its molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and pH 6, thermal stability of 70°C for 40 min, and salt concentration of 1 M with Km and Vmax of 6.58 mg/ml and 21.93 μmol/ml/min, respectively. The α-amylase was improved by adding Cu+2, Zn+2, and Fe+2 . Increased production of α-amylase enzyme by S. rochei KR108310 leads to production of significant industrial products.Amylase producing actinobacteria were isolated and characterized from terrestrial environment. There are a limited number of reports investigating the marine environment; hence, in the present study, four marine enzymes were tested for their amylase production ability. On starch agar plates, the  Streptomyces species are the most essential industrially useful organism because of their ability to produce multiple enzymes , 30α-amyα-amylase protein (presented in a specific activity) in terms of t-value [P value, and confidence level. The level of significance  was resolved using Student's t-test. Every single impact t-test assesses the probability that finding the observed effect was pure chance. If this is highly unlikely, then the effect is thought to be caused by the variable when it is below the accepted level, such as 5%. The confidence level reflects a percentage of the P value. Activities were assessed using the Microsoft Excel solver add-in program. Each response was simultaneously visualized in three-dimensional graphics created using STATISTICA 10.0 software for the four largest independent factors.Numerous straight relapses were made utilizing Microsoft Excel predictions to determine the significance of the  t-value , P valueExperimentally, the optimal conditions were verified by optimization experiments. The predictions were examined and compared to the basic conditions, near-optimal conditions, and conditions different from the optimum levels of the independent variables.α-Amylase was purified from S. rochei HMM 13 using various steps, including precipitation of ammonium sulfate, dialysis, and Sephadex G-50 [dex G-50 . All purdex G-50 . The ultdex G-50 .S. rochei HMM 13 α-amylases was tested under standard assay conditions. The temperature effect was determined on sterilized α-amylase by incubating the α-amylase activity at a temperature extending from 30°C to 80°C utilizing phosphate buffer for 4 h and measuring activity as already described [α-amylase was determined when the enzyme was incubated at 55°C, 65°C, and 70°C for 4 h. Enzyme activity was determined at regular time intervals of 20 min. Kinetic α-amylase activity parameters were calculated at soluble starch concentrations of 0.1 to 1.2 g/l at a pH of 6 and 65°C for 30 min [α-amylase activity was examined by activity at various NaCl concentrations (0.2–2.1 M) in phosphate buffer with preincubation of an enzyme at 65°C and a pH of 6 for 30 min under standard conditions. Metal particles  were contributed to distinguish their impact on α-amylase activity in 0.05 M phosphate buffer at a pH of 6. A measure of 5 ml of metal ion solution and reagents were combined with refined S. rochei HMM 13 α-amylases. This mixture was kept under 65°C for 30 min, and the enzyme activity was measured as depicted by [The most active peak of the purified enzyme is used for characterization. In this step, nine ml of purified enzyme was used to study pH, temperature, thermal stability, kinetic study, salinity, and metal effect on enzyme activity. The pH (3–10) of purified escribed . Thermalr 30 min . Double r 30 min . Optimumicted by .α-amylase is produced from isolated marine actinomycetes on culture plates. Among the actinomycete isolates, the Streptomyces rochei strain HMM 13 was very active on starch agar plates with iodine as a detector (24 mm) when compared to other isolates , S. rochei strain (HMM 13), S. parvus strain (8), and Streptomyces sp. strain (M12). The most significant effect of the four isolates was found for S. rochei HMM 13. The t-test showed that the most critical independent variables influencing α-amylase enzyme development were starch, K2HPO4, pH, and temperature. Consequently, specific activities were detected  level of starch, casein, and K2HPO4, in addition to a negative level (-) of KNO3, supported production by S. griseorubens. However, there was a positive (+) level of starch, KNO3, and MgSO4 in addition to the negative level of pH corroborative enzyme production by S. parvus. Finally, the positive (+) level of starch and negative level (-) of K2HPO4, pH, and temperature supported enzyme production by Streptomyces sp. Additionally, the t-value in The Plackett-Burman design was used to assess the critical impact of utilizing starch casein agar medium components for the generation of detected . The pridetected . Based oS. griseorubens MMH9, S. rochei HMM 13, S. parvus (8), and Streptomyces sp. M12 in verified medium adjusted to a pH of six for seven days resulted in a 1.7, 9.8, 7.7, and 3.12-fold increment in the specific activity measurement compared to the baseline conditions  suggested by the Plackett-Burman design were used to investigate the optimum response region for α-amylase production by S. rochei HMM 13 at three levels  in the BBD , K2HPO4 (X2), pH (X3), and temperature (X4) were effective within the concentration ranges investigated and under the present experimental conditions. These figures suggest that increasing the starch concentration to 19 g/l at a high level of temperature will promote the production of α-amylase enzyme. However, a higher level of enzyme production was accomplished with diminished K2HPO4 concentration.At the model level, the correlation measures for estimating the regression equation are the multiple correlation coefficient α-amylase enzyme by isolate S. rochei HMM 13, the medium formula should be kept at 30.6°C for seven days and formulated as follows (g/l): starch 19 g, KNO3 3 g, casein 0.5 g, MgSO4 0.75 g, FeSO4 0.001 g, K2HPO4 0.2 g, CaCO3 0.1 g, agar 18 g, and 50/50 DW. An investigative study was conducted to compare the anticipated optimal levels of autonomous factors and fundamental conditions. Development of S. rochei HMM 13 within the confirmed medium balanced to pH 8 for seven days resulted in a 12.22-fold increment within the specific activity estimation compared to basal conditions  of purified α-amylase with maximum strength and activity at a pH of 6  and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively  with a maximum pH of 8.0 (118.1 ± 2.3). The optimal pH and α-amylase activity temperature vary from one species to species [α-amylase in submerged fermentation was 40°C (123.12.1 U/ml). Starch improved the production of α-amylase among carbon sources. There was lower α-amylase activity in the other carbon sources chosen [Streptomyces rochei biomass yields and indicated the best conditions to establish a cost-efficient approach to enhance α-amylase production through variables optimized using BBD. Therefore, RSM can be a useful tool for enzyme development by optimizing the various independent factors involved in the fermentation method using the fewest number of experiments [The diversity of marine actinomycetes is important in many areas of science and medicine . They arTSS 1001 , 42. Inirsicolor . On the rsicolor . Studiesrecorded . They ap 23.96 U . Yahya elum size . BBD is lum size . BBD wasobtained . Our ress chosen . The staeriments .α-Amylase is extremely selective, specific, and able to catalyze industrial reactions at low temperature and pressure. Particular α-amylase may be specific and selective, but if the contaminant enzymes have opposite (or just different) properties, this may reduce the apparent performance of the prepared biocatalyst. Therefore, obtaining pure enzymes is very important to participate in various industrial reactions, even if these enzymes are in small quantities [Purified amylase acts as a natural niche as an industrial biocatalyst. antities , 49.α-amylase purification utilizing S. rochei are summarized by ammonium sulfate which is the foremost stable agent for partial filtration [α-amylase was eluted with a relatively low molecular weight one which was capable of entering the pores easily. The use of an appropriate size of gel particles is the key to the purification process [α-amylase whose molecular weight range is from 35 kDa to 55 kDa. Chromatographic methods for protein purification are the most common methods after crude dialysis and partial precipitation [α-amylase immobilization methods are in agreement with other α-amylase immobilization studies [Bacillus amyloliquefaciens; the adsorption process of amylase referred to the weak binding of α-amylase molecules [α-Amylases from actinomycetes have a pH optima in the alkaline to a neutral range [α-Amylase was found to be profoundly dynamic at 65°C. Comparable outcomes were found in another study [α-amylase from S. rochei is thermostable. Several previous studies have detailed that amylase from Streptomyces sp. MSC702 and S. avermitilis 5981 were steady at 60°C and 50°C, respectively [μmol/ml/min, respectively, which is close to the Km of α-amylase produced by S. fragilis DA7-7 by 6.24 mg/ml. Vmax in the current study is higher than Vmax in the study of Nithya and others by 8.36 μmol/ml/min [α-amylase by activity 22.71 U/ml. Previous studies of Anand et al. and Rathore et al. demonstrated that α-amylase from Streptomyces sp. SNAJSM6 and N. dassonvillei KaS11 was steady at 3% and 9% concentrations of NaCl, respectively [+2, Zn+2, and Fe+2 have positive adding metals for increasing α-amylase activity by 152.21%, 207.24%, and 111.89%, respectively, as the result reported by Du et al. [Enzyme partial purification of proteins is chromatographically filtered after crude separation by precipitation and membrane separation . The resltration . The mos process . Therefopitation . The eff studies . The effolecules , 55. Chaal range , which der study , in whicectively , 37. Theectively , 57. Finu et al.  and Asghα-amylase enzyme production by marine S. rochei KR108310 strain HMM 13. To maximize enzyme production, the most effective variables were further adjusted using the Box-Behnken design. The produced enzyme was purified and characterized, being more active at higher temperature and pH, as well as improved by the addition of different metal particles. In general, the characteristics of the pure α-amylase enzyme generated by S. rochei KR108310 have been investigated, making it a suitable candidate for a wide range of applications.The current work used the Plackett-Burman statistical experimental strategy to investigate the major parameters influencing


Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaCA–04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA–04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA–04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA–04 protein with high YP/S and PHB productivity. Soluble phaCA–04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA–04 from pColdTF-phaCABA–04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCABA–04 enhanced soluble protein formation to approximately 3.09−4.1 times higher than that from pColdI-phaCABA–04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCABA–04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from  The global environmental concern regarding microplastics in the marine environment as contaminants with significant impacts on animal and human health has led to a call for national and international policies from more than 60 countries to ban or place a levy on single-use plastics . RenowneCupriavidus necator H16 , is capable of accumulating polyhydroxybytyrate (PHB) at over 80% of the CDM. PHA accumulation is tightly regulated by imbalanced growth conditions with excess carbon but limited nitrogen (phaA), NADPH-dependent acetoacetyl-CoA reductase (phaB), and PHA synthase (phaC), and these three genes are sufficient for the production of PHB in non-PHA-producing bacteria at more than 90% of the CDM when heterologously expressed in Escherichia coli . The best known industrial PHA producer, nitrogen . One of nitrogen . Therefonitrogen . Ordinarhia coli . It has hia coli .C. necator H16 (PhaCH16) is a Class I PhaC and is one of the most widely studied PHA synthases. It has a molecular weight of approximately 64 kDa (589 amino acids) and is located as the first gene in the PHA biosynthetic phaCAB operon, followed by PhaA and PhaB (w) of PHB synthesized by wild-type bacteria is generally in the range of 0.1–2.0 × 106 Da. When recombinant PhaCH16 was overexpressed in E. coli, most of the protein formed insoluble inclusion bodies due to its low aqueous solubility , acetoacetyl-CoA reductase  and PHB synthase . Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference was in the amino acid residue situated at position 122, which in phaCA–04 was proline but in C. necator H16 was leucine. The total amino acid sequences of phaAA–04 and phaBA–04 were 100% matched with those of C. necator H16  [P(3HB-co-4HB)] copolymer under a C/N ratio of 20 (co-3-hydroxyvaterate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer , a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h). Finally, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04, leading the PHB production of 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the cell dry mass (CDM) of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) in LB medium.In a previous study, we reported the generation of the  ability . Designeator H16 . Notablyio of 20 , as wellrpolymer . In the phaCABA–04 genes from the isolated C. necator strain A-04 in pColdI  and pColdTF  chaperone, N-terminal 6His-fusion protein). The reason for choosing pColdI and pColdTF is that there have been few reports on utilizing cold inducible promoter for PHB production. Therefore, the obtained results will prove that if phaC solubility is enhanced, this outcome would finally result in enhancing PHB production. It has been well known that CspA was originally found as the major cold-shock protein in E. coli, consisting of 70-amino-acid residues. CspA forms a β-barrel structure with five anti-parallel β-strands and functions as an RNA chaperone. Its transient induction upon cold shock is regulated at the level of transcription, mRNA stability and translation , which facilitates co-translational protein folding, thus, reducing the chances of forming misfolded and insoluble proteins -inducible vector, N-terminal GST fusion protein], pBAD/Thio-TOPO  (phaCABA–04 biosynthesis genes with native promoter of phaCA–04) and transformed into E. coli JM109   and pUC1li JM109 . Next, tE. coli strains and plasmids used in this study are listed in C. necator strain A-04  medium supplemented with 100 μg/L ampicillin. The LB medium contained (per liter) 10 g of tryptone , 5 g of yeast extract  and 10 g of NaCl . Stock cultures were maintained at –80°C in a 15% glycerol solution. The experiments were performed in a biosafety level 1 laboratory and by researchers and investigators who had undergone biosafety training.The ain A-04  was usedphaCABA–04 operon PHB biosynthetic genes from C. necator A-04 were PCR-amplified using the following pair of primers: forward primer 5′-ATGGATCC CTCGAGATGGCGACCGGCAAAG-3′ (the XhoI site is underlined) and reverse primer 5′-GTGAATTCAAGCTT TCAGCCCATATGCAGGCC-3′ (the HindIII site is underlined). Primers were designed based on accession numbers FJ897463, FJ897461, and FJ897462. The blunted PCR product was purified and subcloned into pBluescript SK-  linearized by SmaI. The recombinant plasmid digested with XhoI and HindIII was cloned into cold-shock-inducible pColdI and pColdTF vectors  at the XhoI and HindIII restriction sites, yielding pColdI-phaCABA–04 and pColdTF-phaCABA–04, respectively. For the plasmid pGEX-6P-1-phaCABA–04, the phaCABA–04 operon was amplified by the primers pGEX-F and pGEX-R , yielding pUC19-nativeP-phaCABA–04. PCRs were performed using Q5® High-Fidelity DNA Polymerase . E. coli JM109 was used as a host for cloning and PHB production. The accuracy of the constructed plasmid was verified by the corresponding restriction enzyme and sequencing.The d pGEX-R . The 3,8phaCABA–04 and pColdTF-phaCABA–04 with the entire phaCABA–04 operon were transformed into E. coli JM109 by the heat shock method (E. coli JM109 cells transformed with pColdI-phaCABA–04 or pColdTF-phaCABA–04 were grown in LB medium containing ampicillin (100 μg/mL) on a rotary incubator shaker  at 37°C and 200 rpm for 24 h. The overnight seed culture was inoculated into fresh LB medium (5% v/v inoculum) containing 100 μg/L ampicillin and 20 g/L glucose prior to induction with temperature and IPTG using three separate induction methods (Expression vectors named pColdI-k method . Shake f methods .600) reached 0.5, 1.3, 2.1, and 2.4. Next, the cultivation temperature was reduced from 37°C to 15°C for 30 min. The expression of the phaCAB operon was induced by the addition of 0.5 mM IPTG, and cultivation was continued at 15°C for an additional 24 h.For the synthesis of PHB using the conventional induction method, the procedure was performed according to the user manual . The culture was incubated at 37°C and 200 rpm until the optical density at 600 nm  of IPTG, and the cultivation was maintained at 37°C for 24 h.For the synthesis of PHB using the short-induction method developed in this study, the culture was incubated at 37°C and 200 rpm until the OD600 reached 0.5. Then, 0.5 mM IPTG was added to the culture and the temperature was reduced from 37°C to 15°C for 24 h. The induced cells were harvested by centrifugation, the medium was discarded, and the cells were resuspended in an equal volume of fresh LB medium. Then, the induced cells at 1, 5, or 10% (v/v) were transferred into fresh LB medium supplemented with 100 μg/L ampicillin and 20 g/L glucose and incubated at 37°C and 200 rpm for 24 h.For the synthesis of PHB using the preinduction method developed in this study, the culture was incubated at 37°C and 200 rpm until the ODE. coli JM109 (pColdI-phaCABA–04) and E. coli JM109 (pColdTF-phaCABA–04) were induced to produce PHB using the conventional induction method and short-induction method. The effect of GST (the hydrophilic fusion protein) and the tac promoter on PHB production was investigated using E. coli JM109 (pGEX-6P-1-phaCABA–04), which was induced by the addition of IPTG (0.5 mM). The effect of the araBAD promoter and N-terminal thioredoxin fusion protein together with the C-terminal 6His-fusion protein on PhaC and PHB production was examined by inducing E. coli JM109 (pBAD/Thio-TOPO-phaCABA–04) with arabinose (1% w/v). E. coli JM109 (pUC19-nativeP-phaCABA–04), which exhibits expression from native promoter without addition of IPTG, was used as a control strain. All of these comparison experiments were performed at 15 or 37°C for 48 h. The crude glycerol used in this study was obtained from biodiesel industries belong to Bangchak Corporation Public Company Limited, a petroleum and energy conglomerate in Thailand. The content of glycerol was 80% w/v.For comparison of the effect of phaC expression on PHB production under various types of promoters, fusion proteins and chaperones, shake flask experiments were performed in 250-mL Erlenmeyer flasks containing 50 mL of LB medium containing ampicillin (100 μg/mL) on a rotary incubator shaker at 37°C and 200 rpm for 24 h. For PHB production, overnight cultures in LB medium (1 mL) were transferred into fresh LB medium supplemented with glucose (20 g/L) and ampicillin (100 μg/mL). Recombinant 600 of 0.5 was obtained, the cultivation temperature was reduced from 37 to 15°C for 30 min. Next, IPTG was added to the culture at a final concentration of 0.5 mM. After IPTG addition, the cultivation temperature was shifted from 15 to 37°C and maintained at 37°C for 48 h. Culture samples were collected at 6 h intervals for 48 h.A preculture was prepared in 500-mL Erlenmeyer flasks containing 100 mL of LB medium and grown on a rotary shaker at 37°C at 200 rpm for 24 h. The preculture was inoculated into a 5-L bioreactor  containing 2 L of LB medium supplemented with 100 μg/L ampicillin and 20 g/L glucose at an inoculation volume of 5% (v/v). The agitation speed and the air flow rate were 500 rpm and 1 mL/min, respectively. After an ODCell growth was monitored by the CDM, which was determined by filtering 5 mL of the culture broth through preweighed cellulose nitrate membrane filters . The filters were dried at 80°C for 2 days and stored in desiccators. The net biomass was defined as the residual cell mass (RCM), which was calculated by subtracting the amount of PHB from the CDM. The PHB in dried cells was methyl-esterified using a mixture of chloroform and 3% (v/v) methanol-sulfuric acid (1:1 v/v) . The resE. coli cells were cultured with and without induction. Cells were collected by centrifugation at 17,000 × g and 4°C for 30 min. Cell pellets were resuspended in 100 mM Tris-HCl (pH 8.0) and normalized to an OD600 of 2.0. Total proteins were extracted from cells by using a sonicator . The lysis mixture was then centrifuged at 17,000 × g at 4°C for 30 min. The protein concentration in the supernatant (soluble protein) was estimated by the Bradford method using a Bio-Rad protein assay kit , and bovine serum albumin was used as a standard. Thirty micrograms of total protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels under reducing conditions and electrophoresed at 80 V for 10 min followed by 140 V for 60 min. For the western blot analysis, the protein from SDS-PAGE was then transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry blotting system  at 150 mA for 40 min. The 6His tag was detected by a mouse anti-His antibody  and an HRP-conjugated goat anti-mouse IgG as the primary and secondary antibodies, respectively. Color development was performed using a Mouse IgG DAB Chromogenic Reagent Kit  according to the manufacturer’s instructions.Recombinant 2PO4, 300 mM NaCl and pH 8.0). Lysozyme  and Benzonase® endonuclease  were added to concentrations of 0.2 mg/mL and 20 U/mL, respectively. The cells were ruptured by ultrasonic homogenizer . The amplitude was set to 40% . The lysate was clarified by centrifugation at 16, 100 × g for 20 min and the supernatant was collected. Protino@® Ni-IDA 1000 His-Tag Protein purification columns  were pre-equilibrated with four bed volumes of 1X LEW buffer and allowed to drain by gravity. The cleared supernatant with 2 mg of total protein was loaded onto a pre-equilibrated column and washed with four bed volumes of 1X LEW buffer containing 20 mM imidazole. Finally, the polyhistidine-tagged protein was eluted with elution buffer . Each fraction was analyzed by running 10 μL of eluate on SDS-PAGE and quantified by Bradford protein assay. To ensure accurate quantitation of yields, the lysate flow-through was collected for detection of unbound product by SDS-PAGE analysis.The cell pellet (from 50 mL culture) was resuspended in 1 ml of lysis-equilibration-wash buffer  membrane filter . The temperature was 40°C and the flow rate was 0.8 mL/min. A standard curve was determined for polystyrene with low polydispersity in the same conditions for the molecular weight 1.26 × 103, 3.39 × 103, 1.30 × 104, 5.22 × 104, 2.19 × 105, 7.29 × 105, 2.33 × 106, and 7.45 × 106. The weight-average molecular weight (Mw) and the number-average molecular weight (Mn) were determined by gel permeation chromatography (GPC) and the polydispersity index (PDI) was calculated as the ratio The molecular weight was determined by Gel Permeation Chromatography  with a 10A refractive index detector and two Shodex columns  belong to Associate Professor Takeharu Tsuge’s laboratory at Department of Materials Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, Yokohama, Japan. Polymer was dissolved in 0.1% (w/v) chloroform and filtered through a 0.45 μm low protein binding DuraporePHB films were prepared according to the ASTM: D882-91 protocol. The PHB films were prepared from chloroform solutions of the polyesters using conventional solvent-casting techniques and a glass tray , comparing E. coli JM109 (pColdI-phaCABA–04) and E. coli JM109 (pColdTF-phaCABA–04), are summarized in phaCABA–04.Next, a short-induction method was investigated in this study with the aim of accelerating growth and PHB production and attaining higher productivity than that afforded by the conventional induction method. First, conditions were optimized by varying the ODThe optimal short-induction temperature was investigated in a range between 15°C and 37°C for 30 min before increasing the temperature to 37°C for 24 h to confirm that the high PHB productivity resulting in this study is a result of the cold-shock cspA promoter and that 15°C is the optimal induction temperature. 600 reached 0.5, IPTG was added at 0.5 mM into the culture, and the temperature was reduced from 37 to 15°C. Then, cultivation was performed for 24 h to allow full expression of the phaCABA–04 protein. Concurrently, the effect of inoculum size  of induced cells was investigated under the preinduction conditions. The results are shown in comparison with those of the conventional induction and short-induction methods (E. coli JM109 (pColdI-phaCABA–04) with an inoculum size of 0.5% (v/v) and cultivated until the OD600 reached 0.5 (2 h) before induction with 0.5 mM IPTG was selected to investigate the effect of induction temperature in the subsequent experiment.We also investigated a preinduction strategy to enhance PHB productivity by extending the PHB production phase at 37°C for an additional 24 h after conventional induction. When the OD methods . The pre600 of 0.5, cold shock induced with 15°C for a short time, 30 min, and the addition of 0.5 mM IPTG. These conditions were selected as optimal parameters for scaling up production in a 5-L fermenter. The comparison between pColdI-phaCABA–04 and pColdTF-phaCABA–04 in a 5-L fermenter by the short-induction method was performed because the ratio of the soluble fraction and inclusion bodies of the phaCA–04 protein may affect PHB productivity and molecular weight distribution as reported by Altogether, for flask cultivation, the optimal conditions were the short-induction method using an inoculum of 0.5% (v/v) in a culture with an ODE. coli JM109 (pColdI-phaCABA–04) (E. coli JM109 (pColdTF-phaCABA–04)  was a more effective PHB producer than the other strain. A PHB content of 89.8 ± 2.3% (w/w), PHB production of 7.9 ± 0.7 g/L, CDM production of 8.8 ± 0.5 g/L, YP/S value of 0.38 g PHB/g glucose and productivity of 0.43 g PHB/(L.h) were the maximum values obtained using pColdTF-phaCABA–04, whereas a PHB content of 80.6 ± 2.1% (w/w), PHB production of 5.8 ± 0.1 g/L, CDM production of 7.2 ± 0.3 g/L, YP/S value of 0.32 g PHB/g glucose and productivity of 0.24 g PHB/(L.h) were attained using pColdI-phaCABA–04. The phaCA–04 protein produced by pColdTF-phaCABA–04 was more stable and longer lasting   and E. cCABA–04) . The sol lasting  than thativation . TherefoE. coli JM109 (pColdI-phaCABA–04) had an Mw of 8.17 × 105 Da, an Mn of 1.97 × 105 Da and a PDI of 4.1, whereas the PHB from E. coli JM109 (pColdTF-phaCABA–04) had an Mw of 2.6 × 105 Da, an Mn of 0.95 × 105 Da and a PDI 2.8, when glucose was used as a carbon source. However, the PHB from E. coli JM109 (pColdI-phaCABA–04) obtained using crude glycerol had the lowest Mw of 2.42 × 105 Da, an Mn of 0.89 × 105 Da and a PDI of 2.92. Interestingly, the PHB from E. coli JM109 (pUC19-nativeP-phaCABA–04) obtained using crude glycerol showed the highest Mw of 1.1 × 106 Da, an Mn of 2.6 × 105 Da and a PDI of 4.1. The melting temperature, Tm, of all the PHB film samples produced in this study was in the range of 165−178°C  possessed the highest values of 5465 and 56.2 MPa, respectively. C. necator strain A-04, E. coli JM109 (pColdI-phaCABA–04) and E. coli JM109 (pColdTF-phaCABA–04). The PHB films prepared by the film casting technique and produced from E. coli JM109 (pColdTF-phaCABA–04) showed a soft morphology with high transparency, which was different from the properties of the other PHB films. The PHB films were also subjected to 1H-NMR and 13C-NMR analyses and showed only chemical shifts of the PHB structure.The PHB thin films were subjected to thermal analysis by DSC, molecular weight determination by GPC and mechanical property analysis by a universal testing machine as per the ASTM: D882-91 protocol . The PHB65−178°C , and theof 1−4°C . The YouH16, as a type I synthase and the most intensively studied of these proteins, preferentially catalyzes the polymerization of short-chain (R)-hydroxyalkanoic acids (4 to 6 carbon atoms), particularly the conversion of (R)-3-hydroxybutyrate-coenzyme A (3HBCoA) to poly(3-hydroxybutyrate) (PHB) . Thus, C. necator strain A-04 can be considered as one of the promising candidates for PHA production. However, the PHA productivity of C. necator strain A-04 still does not meet the requirement for industrial production. Thus, our objective was to investigate the ability of the phaCABA–04 gene operon when heterologously expressed in recombinant E. coli. We initially aimed to use the pColdI and pColdTF expression systems to address the challenges of soluble and functional phaCA–04 expression in E. coli JM109 from glucose as a carbon source for PHB production in a 5-L fermenter and evaluate its ability to use crude glycerol to attain low cost PHB production.It has been reported that PhaCe) (PHB) . In facttsZ gene . UltrahitsZ gene . Beyond n source , 2010. IA–04 expression in E. coli JM109 by using the cold-shock cspA promoter and TF. His-tagged phaCA–04 was overexpressed by pColdI, but most of the protein was present in insoluble form, with significant aggregation resulting in smear bands  PHA synthases were differentiated distinctly based on the highest level of PHB accumulation at different temperatures. They demonstrated that the parental enzymes PhaCH16 had optimal temperature of 37°C  cycle  for PHB production from glucose. They reported that YP/S of E. coli strain XL1-Blue (pSYL105) grown in LB medium containing 20 g/L glucose was as high as 0.369 g PHB/g glucose whereas JM109 provided 0.299 g PHB/g glucose. Thus, the YP/S obtained in this study was comparable. In the fed-batch cultivation studies, E. coli XL1-Blue (pSYL107) in a 50-l fermentor and attained YP/S of 0.28 g of PHB/g of glucose . They mentioned that a slightly lower PHB yield on glucose in a defined medium than in LB medium (0.37 g of PHB/g of glucose) because glucose was converted to CO2 in a defined medium more than in LB medium  were obtained in 51 h, resulting in the PHB productivity of 2.0 g/L/h. This is the highest PHB concentration obtained by employing recombinant E. coli containing the phaCABH16 biosynthesis genes and the E. coli ftsZ gene. In our previous report, we performed fed-batch cultivations by pH-stat control in a 5-L fermenter using E. coli strain XL1-Blue harboring pColdTF-phaCABA–04, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) in define medium. To investigate the possibility of improving the PHB yield and productivity, we are now carrying out fed-batch cultures under various conditions including oxygen limitation during PHB synthesis phase as it may be possible to enhance the PHB synthesis rate by increasing the acetyl-CoA flux into the PHB biosynthetic pathway and reducing its flux into the tricarboxylic acid cycle  cycle . Acetyl-ding PHB . The mairogenase . Throughuctivity . In addiuctivity , leadinguctivity . Therefouctivity . It has  acetate . With thB medium . Next, tid cycle .w of PHB produced from pColdTF-phaCABA–04, for which soluble phaCA–04 was 4.1 times higher than pColdI-phaCABA–04 was lower than that from pColdI-phaCABA–04. w and Mn of PHB produced by pColdTF were lower than those of pColdI-phaCABA–04 and pUC19-nativeP-phaCABA–04. In this case, TF increases PhaC production and its Mw decreases due to the presence of more active PhaC. To achieve low-cost production, crude glycerol containing 80% glycerol provided by Bangchak Corporation Public Company Limited as a byproduct from biodiesel production, was used as a carbon source to produce PHB using pColdI-phaCABA–04 and pUC19-nativeP-phaCABA–04. With E. coli JM109 (pColdI-phaCABA–04), PHB produced from crude glycerol had an Mw of 2.42 × 105 Da and an Mn of 0.89 × 105 Da with a PDI of 2.92, whereas those results from glucose were Mw of 8.41 × 105 Da and an Mn of 2.03 × 105 Da with a PDI of 4.14. The crude glycerol caused the termination step involves a chain transfer (CT) reaction in which the polymer chain is transferred to the crude glycerol (CT agent) in this case , a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L⋅h) using pColdTF-phaCABA–04. The PHB from pColdTF-phaCABA–04 had Mw 5.79 × 105 Da, Mn 1.86 × 105 Da and PDI 3.11 and the film exhibited high optical transparency with typical melting temperature and mechanical properties.This study revealed that the cspA promoter in a cold-inducible vector can enhance total PhaChttps://www.ncbi.nlm.nih.gov/genbank/, FJ897463; https://www.ncbi.nlm.nih.gov/genbank/, FJ897461; and https://www.ncbi.nlm.nih.gov/genbank/, FJ897462.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: TB performed the experiments and discussed the results. RW-S provided guidance for the experimental design and discussed the results. KH provided suggestions for the experimental design and discussed the results. SCN provided guidance and suggestions for the experimental design, discussed the results, and wrote and revised the manuscript. All the authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.