While primarily found in endo-lysosomal compartments, the cysteine protease legumain can also translocate to the cell surface if stabilized by the interaction with the RGD-dependent integrin receptor αVβ3. Previously, it has been shown that legumain expression is inversely related to BDNF-TrkB activity. Here we show that legumain can conversely act on TrkB-BDNF by processing the C-terminal linker region of the TrkB ectodomain in vitro. Importantly, when in complex with BDNF, TrkB was not cleaved by legumain. Legumain-processed TrkB was still able to bind BDNF, suggesting a potential scavenger function of soluble TrkB towards BDNF. The work thus presents another mechanistic link explaining the reciprocal TrkB signaling and δ-secretase activity of legumain, with relevance for neurodegeneration. Cell signaling allows the transfer of information between cells to coordinate different tissues and cell types ,2.Of particular relevance are neuronal receptors such as the enzyme-coupled receptors p75NTR and Tyrosine-receptor-kinase B (TrkB), which transduce apoptosis and survival signals, respectively. These receptors work in concert with each other . By thesKnown physiologic regulators of TrkB are the neurotrophins BDNF and NT4, which specifically bind TrkB and induce its dimerization . AdditioCombining the above-mentioned information led us to explore the possibility of TrkB being processed by legumain in its ectodomain and its potential impact on TrkB binding to its specific binding partner BDNF. Here we identify legumain cleavage sites in the ectodomain of TrkB and show that this processing can create soluble TrkB isoforms with the ability to bind BDNF as well as the pro-form of BDNF. Additionally, we also show that the complex formation of TrkB and BDNF prevents the processing by legumain.302-Cys345 is predominantly formed, To study the extracellular TrkB interaction with relevant partner molecules, we produced the ectodomain of TrkB (Ser29-Glu428) in HEK293S cells, carrying a C-terminal His6-tag for purification and concentration, E. coli by in vitro folding from inclusion bodies, 143) with cysteine-4 (Cys210), cysteine-2 (Cys188) with cysteine-5 (Cys239), and cysteine-3 (Cys199) with cysteine-6 (Cys241). The first disulfide bond of Cys143-Cys210 could be directly detected, whereas the second and third bonds, Cys188-Cys239 and Cys199-Cys241, could not be separated but detected in a common peptidic fragment. Proteolytic cleavage proved impossible and reflected the sequential and spatial proximity of cysteine 5 and 6, which are located in the tripeptide Cys239-Val240-Cys241. The used protease, Trypsin, does not cleave after valine. The production protocol resulted in approximately 300 µg of pure protein from 1 g inclusion bodies for BDNF. Legumain was produced as previously described  were injected using in-line split-loop mode and separated at a flow rate of 200 µL.min−1 and a column oven temperature of 70 °C applying the following linear gradient: 5.0% B for 10.0 min, 5.0–40.0% B for 20 min, 40.0–80.0% B for 5.0 min, 80.0% B for 5.0 min and 5.0% B for 20 min. UV detection was carried out at 214 nm using a 1.4 µL flow cell.® mass spectrometer equipped with an Ion Max™ source with heated electrospray ionization (HESI) probe, both from Thermo Fisher Scientific , and an MXT715-000-MX Series II Switching Valve . Mass spectrometric data were acquired between minute 10.0 and 55.0 in an m/z range of 500–3000 with instrument settings as specified earlier [m/z 200, and legumain-digested Ig2 was analyzed at a resolution of 140,000 at m/z 200. MS/MS of intact and legumain-digested Ig2 was carried out applying all ion fragmentation (AIF) in the higher-energy collisional dissociation (HCD) cell at normalized collision energy (NCE) settings of 22.0 within a scan range of m/z 400–2500 and a resolution setting of 140,000 at m/z 200. Peptide and disulfide mapping was conducted using data-dependent MS/MS. Each scan cycle consisted of a full scan at a scan range of m/z 400–2000 with an AGC target of 3 × 106, a maximum injection time of 100 ms and a resolution setting of 70,000, followed by 5 data-dependent HCD scans at 30 NCE with an AGC target of 5 × 105, a maximum injection time of 200 ms and a resolution setting of 17,500. The dynamic exclusion was set to 10.0 s.All samples were analyzed on a Thermo Scientific™ QExactive™ benchtop quadrupole-Orbitrap earlier . Intact Isotopically resolved mass spectra were deconvoluted using the Xtract algorithm implemented in the Xcalibur™ software version 3.0.63 . Isotopically unresolved spectra were deconvoluted using the Respect algorithm implemented in Biopharma Finder 1.0 . AIF data were evaluated using the software ProSight Lite v1.4 Build 1.4.6 provided by the Kelleher Research Group ; mass tolerance for annotation of b- and y-fragments was set to 25 ppm. Peptide mapping data were evaluated using Biopharma Finder 1.0.Analysis of proBDNF and tryptic peptides thereof. For intact mass analysis, 300 µg of TCA precipitated proBDNF were dissolved in 175.0 mmol L−1 ammonium acetate. For disulfide mapping, 400 µg of TCA-precipitated proBDNF were dissolved in 100 µL 175.0 mmol L 1 ammonium acetate. Trypsin  was directly added and allowed to digest overnight at 37 °C and 900 rpm. The reaction was stopped by the addition of FA to a final concentration of 1%.−1: 15.0% B for 3.0 min, 15.0–90.0% B for 27.0 min, 90.0–99.0% B for 0.1 min, 99.0% B for 5.0 min, 99.0–5.0% B for 0.1 min, 5.0% B for 5.0 min.Intact proBDNF analysis was carried out in a Vanquish HPLC system . Five microlitres of the sample were injected and separated during a linear gradient at 60.0 °C and a constant flow rate of 100.0 µL min® RSLC column . One microlitre of the sample was injected in full-loop mode. proBDNF-peptides were separated at 1.2 µL min 1. For peptide analyses, the linear gradient was as follows: 1.0% B for 5.0 min, 1.0–30% B for 30.0 min, 30.0–60.0% B for 5.0 min, 99.0% B for 5.0 min, 1.0% B for 10.0 min.Tryptic peptides were analyzed on a nano-HPLC system  with an autosampler set to 4.0 °C and column oven to 50 °C. proBDNF-derived peptides were separated on an Acclaim PepMapm/z range of 1000–3500 at a resolution of 140,000 at m/z 200 using the following instrument settings: AGC target 3 × 106, maximum injection time of 150 ms, 10 microscans, capillary temperature 300 °C, and S-lens RF level 50. Tryptic peptides of proBDNF were ionized on a nano-ESI source under positive polarity at 1.5 kV. Peptides were acquired in full MS mode throughout the chromatographic run (0.0–55.0 min) within a scan range of m/z 400–3000 at a resolution of 70,000 at m/z 200 with an AGC of 3 × 106, maximum injection time of 100 ms. The capillary temperature was set to 250 °C, S-lens RF level to 60. For fragmentation, a top 10 method was utilized within a scan range of 200–2000 m/z at a resolution of 17,500 at 200 m/z with an AGC of 1 × 105 and a maximum injection time of 50 ms. The normalized collision energy was set to 28.0% and the dynamic exclusion to 10.0 s. All used HPLC-MS parameters are summarized in Intact proBDNF, as well as tryptic peptides thereof, were analyzed on a benchtop Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer . Intact proteins were sprayed from a HESI source in positive ion mode. Ion source settings for proBDNF were as follows: source heater temperature 150 °C, spray voltage 4.5 kV, sheath, auxiliary, and spare gas flow 10, 8, and 0 arbitrary units, respectively. proBDNF was analyzed in full MS mode throughout the chromatographic run (0.0–50.0 min) over an ® algorithm for isotopically resolved spectra. Detailed settings for evaluation parameters can be found in For disulfide mapping, simulated isotope patterns were generated in Thermo Xcalibur Qual Browser software (v.4.2.28.14) with the following settings: the chemical formula was calculated in excel, 100% hydrogen adduct, most abundant charge state 2–4, output style profile , resolution 70,000, valley FWHM . Extracted ion current chromatograms were calculated by Thermo Xcalibur Qual Browser software using the following settings: enabled smoothing using Boxcar for 7 points, baseline subtraction enabled, mass tolerance 10 ppm. Peptide data was evaluated in Byonics . For proBDNF-peptides, lysine and arginine were defined as cleavage sites allowing 2 missed cleavages. Parameters for instrument settings were precursor mass tolerance of 20 ppm, fragmentation type CID low energy, and a fragment mass tolerance of 20 ppm. The protein false discovery rate (FDR) was set to 1%. Spectrum output options: charge state 1–6, maximum precursor mass 10,000 Da; protein output options: 2% FDR. Disulfide mapping was activated (−2.015650 Da at cysteine) was activated for proBDNF. The mass spectra of intact proBDNF were deconvoluted using BioPharma Finder 3.0 (Thermo Scientific) using the XtractThe enzymatic activity of legumain was investigated using the peptidic Z-Ala-Ala-fluorescence was measured at 460 nm upon excitation at 380 nm. Processing Asn-7-amino-4-methylcoumarin  substrate. The activity was measured in assay buffers composed of 100 mM citric acid pH 5.5 and 50 mM NaCl or 100 mM Hepes pH 7.0 and 50 mM NaCl supplemented with 50 µM of the substrate. Additionally, the effect of the reducing agent DTT was tested upon the addition of 5 mM DTT to the assay buffers. Reactions were started by the addition of the enzyme at an 8 nM concentration. Assays were carried out at 37 °C in an infinite M200 plate reader . An increase in fluorescence was measured at 460 nm upon excitation at 380 nm.Processing of TrkB ECD and Ig2 with legumain was done by mixing 0.5–1 mg/mL TrkB ECD/Ig2 and 0.007–0.055 mg/mL legumain in cleavage assay buffer  which was pH-fixed to pH 7 or 5.5 with either 1 M HEPES, 1 M Tris pH 7 or 1 M citric acid pH 5.5 . The mixture was incubated for 10–15 min at RT. For MS measurements, legumain was inactivated by adding 1 M Ammonium acetate pH 8.6 at 1:1 ratio of total volume. An exemplary assay with respective ratios and volumes can be found in The TrkB cleavage assay was done as described above and shown in the Processed TrkB ECD was injected into the AKTA FPLC system  with a Superdex 200 10/300 GL column (Cytiva Lifesciences) attached, while for TrkB Ig2, a Superdex 75 10/300 GL was used. Both columns were equilibrated with 1x PBS pH 7.4. Fractions with no visible legumain on SDS-PAGE were pooled and concentrated with a 5 kDa cutoff Amicon .Preparation. To allow complex formation, 47 µL of 0.3 mg/mL BDNF were mixed with 3 µL of ~4 mg/mL TrkB ECD/Ig2 for a total concentration of ~0.28 mg/mL of both in 1x PBS pH 7.4. The mixture was incubated at RT for 10 min. Legumain was diluted in cleavage assay buffer to a stock concentration of 1.875 mg/mL.Cleavage assay. The complex cleavage assay was then mixed together similarly to the cleavage assay described before or as described in Protein labeling. BDNF (20 µM), intact TrkB ECD/Ig2 (20 µM/20 µM), processed TrkB ECD/Ig2 (4.9 µM/15 µM) were labeled with NT-647 RED dye  according to the protocol provided by NanoTemper with the following change: The buffer was not exchanged with the protocols exchange buffer, but all proteins were kept in either 1x PBS pH 7.4 or 20 mM HEPES, 100 mM NaCl, pH 7.5.Incubation. For the following combinations of labeled and unlabeled proteins, each preparation step was the same: labeled intact TrkB ECD/Ig2 (0.65 µM/0.79 µM) with unlabeled BDNF (20.5 µM), labeled processed TrkB ECD/Ig2 (0.25 µM/0.52 µM) with unlabeled BDNF (20.5 µM) and labeled BDNF (1.1 µM) with unlabeled TrkB ECD/Ig2 (20 µM/20 µM). Absorption at 280 and 650 nm was measured for each labeled protein to determine the concentration and degree-of-labeling (DOL) before being diluted 1:67 or 1:100 in 180 µL ice cold MST buffer . Next, 10 µL of unlabeled protein was diluted 1:2 per step in a 16x dilution series with MST buffer at RT. To each 10 µL of the dilution series, 10 µL of diluted labeled protein was added and mixed by pipetting. The mix was incubated in the dark at RT for 10 min and centrifuged for 5 min at 13,000 rpm at 4 °C . Thereafter, each of the 16 dilution steps was loaded into a capillary  and placed into the NT.115-Monolith  thermophoresis device.Measurement and data generation. MST signals were measured with the Monolith NT.115 MST device and recorded with MO.Control Software . Excitation Power was set to 40% for Nano-RED, and MST-Power was set to medium. Acquired data were evaluated and affinities were determined with MO.Affinity Software .


Pseudomonas sp. L3, Pseudomonas sp. P2, Streptomyces sp. T3, and Streptococcus sp. T4) previously isolated from three different agroforestry zones and already screened for wheat growth under pot trial conditions were evaluated on wheat crop under field conditions. Two field experiments were employed; set 1 includes PSB + recommended dose of fertilizers (RDF) and set 2 includes PSB – RDF. In both field experiments, the response of the PSB-treated wheat crop was significantly higher compared to the uninoculated control. In field set 1, an increase of 22% in grain yield (GY), 16% in biological yield (BY), and 10% in grain per spike (GPS) was observed in consortia  treatment, followed by L3 and P2 treatments. Inoculation of PSB mitigates soil P deficiency as it positively influences soil alkaline phosphatase (AP) and soil acid phosphatase (AcP) activity which positively correlated with grain NPK %. The highest grain NPK % was reported in CNS-treated wheat with RDF  and without RDF , respectively. All parameters, including soil enzyme activities, plant agronomic data, and yield data were analyzed by principal component analysis (PCA), resulting in the selection of two PSB strains. The conditions for optimal P solubilization, in L3  and P2 , were obtained through response surface methodology (RSM) modeling. The P solubilizing potential of selected strains at <20°C makes them a suitable candidate for the development of psychrotroph-based P biofertilizers. Low-temperature P solubilization of the PSB strains from agroforestry systems makes them potential biofertilizers for winter crops.It is well-known that phosphate-solubilizing bacteria (PSB) promote crop growth and yield. The information regarding characterization of PSB isolated from agroforestry systems and their impact on wheat crops under field conditions is rarely known. In the present study, we aim to develop psychrotroph-based P biofertilizers, and for that, four PSB strains ( This negatively affects the soil quality and puts an economic burden. Therefore, alternative methods for using P-based fertilizers economically without sacrificing soil quality are required to support sustainable agriculture. Since PSBs mobilize P to plants, which would otherwise remain deposited in the soil, the application of phosphate-solubilizing bacteria (PSB) becomes imperative. A century's worth of agricultural P would be supplied by the bacterial-mediated mobilization of free P from complex ore  can increase wheat crop output  is not only an essential nutrient for plant growth but is also involved in diverse biochemical processes like lipid metabolism and the biosynthesis of cell membranes and nucleic acids . CAMs can be both psychrophilic and psychrotrophic. Pseudomonas sp.-L3 (Accession No. MG966341), Pseudomonas sp.-P2 (Accession No. MG966347), Streptomyces sp.-T3 (Accession No. MG966352), and Streptococcus sp.-T4 (Accession No. MG966353), were isolated from three Dalbergia sissoo Roxb. provenances in North region of Uttarakhand, India, located at Lachhiwala , Pantnagar (29.0369°N latitude and 79.4472°E longitude), and Tanakpur , India  and zinc solubilizing index (ZSI) of four PSB strains were calculated  by adding α-amino naptho sulfonic acid in the treated sample . At 5 DPI, prior to estimating P, the pH of bacterial cultures was monitored.The quantitative inorganic phosphate-solubilizing potential of four PSB strains was assessed in the National Botanical Research Institute's Phosphate (NBRIP) medium containing glucose (10 g), tri-calcium phosphate-TCP (5 g), MgCl2 (5 g), MgSO4.7H2O (0.25 g), KCl (0.20 g), (NH4)SO4 (0.10 g), distilled water 1 (L), and the pH (7.0 ± 0.2). The log phase culture of four PSB strains was inoculated in NBRIP broth medium and incubated at 30 ± 2°C, 120 rpm in an incubator shaker for 5 days. Thereafter, cell-free supernatant was used to measure soluble P via a bacteriological filter (0.2 μM). An equal proportion of filtrate and ethyl acetate were mixed thoroughly for 20 min in a separating funnel, and after vigorous shaking, two layers were developed. The upper (ethyl acetate) layer containing the metabolites was separated, and the extract was concentrated by a rotator evaporator for analysis with FTIR , followed by filtration via permutation and combination  liquid broth culture of four test PSBs and consortia . Field preparation for both experiments was started 1 week before the field setting. The field was plowed, and bunds of 15 cm in height were prepared. Each plot was 2 m long and 2 m wide having a net area of 4 m2. Approximately six lanes per plot were made and leveled carefully after seeds were set.A superior wheat genotype UP 262 was obtained from the seed processing Center (SPC), G.B. Pant University of Agriculture and Technology, Pantnagar, India. The seeds were surface-sterilized with 1% sodium hypochlorite (NaOCl) solution followed by two/three times washing with sterile distilled water. The surface-sterilized seeds were then dried for 1 h in laminar followed by bacterization −1 with field design given in Two sets of the field were prepared. The set 1 (RDF + Treatments) and set 2 (RDF – Treatments) detailed information about the treatments is mentioned in Plant samples were carefully uprooted at 45 and 90 days post-inoculation (DPI) and analyzed for plant vigor parameters. Soil samples were also taken and analyzed for soil physico-chemical properties and soil enzyme parameters.Wheat samples (45 and 90 DPI) were subjected to biometric observation such as plant height, plant fresh weight, and number of tillers per plant. The grain yield (GY), biological yield (BY), 1,000 g wt, and harvest index (HI) were taken at the final harvesting. At final harvesting, the plants were dried for 72 h followed by thrashing. HI of each plot was calculated with the help of the following formulae:For soil health assessment, three soil enzyme activities, e.g., alkaline phosphatase (AP), acid phosphatase (AcP), and fluorescein diacetate (FDA) hydrolysis, were determined to evaluate the impact of PSB inoculation on soil health.2) was added to stop the reaction and Tris buffer was added. The sample was then subjected to centrifugation followed by filtration through Whatman filter paper (0.2 μm) , and 1 mL of 25 mM p-nitrophenyl phosphate (PNP) were added in a test tube and incubated at 37 ± 1°C for 2 h under gentle shaking condition. Calcium chloride  solution was used. Tris buffer of strength 0.1 M (pH −7.4) and TTC solution of 5 mL were added in a 100 mL flask containing 5 g of soil and incubated for 8 h. To stop the reaction, 25 mL of toluene was added followed by centrifugation at 4,000 rpm for 10 min. The supernatant collected from this was filtered, and the absorbance was read at 485 nm  for total bacteria count, Ashby media for total nitrogen fixers, Aleksandrow media for potassium solubilizers, and Pikovskaya agar for P solubilizers, respectively. The soil samples were serial-diluted followed by pour plating, and then, plates were incubated for 2 days at 28 ± 30° C. Thereafter, bacterial colonies were counted  were analyzed before and after the final harvesting of the wheat crop. Soil pH and EC were determined according to the method of Bower and Wilcox  and JackWheat plants of both the field experiments, set 1 and set 2, were harvested, and their seeds were analyzed for N, P, and K content according to the method of Hanway and Heidel  and SubbThe organic phase of PSB strains (L3 and P2) was extracted as mentioned earlier in Section 2.3. The organic phase was further subjected to the direct ESI-MS using a Water Q-ToF micro mass spectrometer.via dimethyl sulfoxide (DMSO) method  of wheat plants were estimated The same procedure was followed for the estimation of carotenoid except that the absorbance of leaf extract was taken at 480 nm  concentration. To run this model, a central composite rotatable design (CCRD) including three variables with four central points was used. RSM model includes a base design that includes both upper and lower limits of the variable along with central points. 3D RSM plots were generated using a mathematical model , in vitro and in vivo field results and further mapped them according to the response shared by them and finally resulted in the selection of potential PSB strains.Principle component analysis was done to evaluate the overall response of All four PSB strains tested positive for inorganic P solubilization. The maximum PSI response was observed in P2 (3.6 cm) followed by L3 (3.4 cm), T3 (2.7 cm), and T4 (2.4 cm). The four PSB strains also tested positive for Zn solubilizing potential, and maximum ZSI was observed in T3 (3 cm) followed by P2 (2.8 cm), T4 (2.6 cm), and L3 (1.8 cm) .cd μgmL−1h−1) followed by P2  were used for recording agronomic traits.e), BY (105.84 ± 0.88f), HI (44.30 ± 0.08d), ET (12.16 ± 0.12de), 1,000 g wt (24.07 ± 0.21d), DMA (34.18 ± 0.18d), and GPS (36 ± 0.14e) being followed by P2 treatment , shoot fresh weight (SFW), root length (RL), and root fresh weight (RFW) was observed in CNS treatment followed by P2 treatment and agronomic response shared by them is significantly higher than the uninoculated control (C) . Overallreatment . In PCA reatment , B. Durireatment . There wreatment .d), BY (86.83 ± 0.14e), HI (34.39 ± 0.25cd), ET (9.6 ± 0.12de), 1,000 g wt (19.96 ± 0.16e), DMA (16.64 ± 0.05cd), and GPS (15 ± 0.03cd) being followed by P2 treatment  . Overallreatment . The PCAreatment , D. Clusreatment . Howeverreatment .f) followed by T4 treatment (666 ± 0.18ef) which is quite significant from uninoculated control (C) soil (466 ± 0.12a μgmL−1h−1). In soil AcP activity, the maximum response was observed in CNS treatment (662.62 ± 0.32e μgmL−1h−1) activity. Soil AcP activity of uninoculated control-C (466 μgmL−1h−1) was found to be significant on the lower side. FDA represents the total soil microbial activity, and among treatments, the maximum response was observed in CNS treatment (48.06 ± 0.014e) followed by P2 (46.64 ± 0.08de μgmL−1h−1) which were significant on the higher side as compared to uninoculated control-C (34.46 ± 0.16a) (f μgmL−1h−1) followed by P2 (632.24 ± 0.26de μgmL−1h−1), and control (C) soil sample was found to be 412.68 ± 0.21a μgmL−1h−1, respectively. In soil AcP activity, the maximum response was observed in CNS-treated soil (645.6 ± 0.18f μgmL−1h−1) and T4 (610 ± 0.22de μgmL−1h−1). In FDA activity in soil field experiment set 2, the maximum response was shown by CNS treatment (38.87 ± 0.13cd) and P2 (38.84 ± 0.18cd μgmL−1h−1)  . In a fimL−1h−1) .4 cells mL−1. At final harvesting in the field experiment set 1, maximum count was observed in soil treated with CNS  which were significantly high as compared to uninoculated treated soil . In a field experiment set 2, a similar pattern was observed in which maximum response was observed in soil treated with CNS  and in uninoculated control   . Alpha dCA-2.54)  and set CA-2.54)  was measCA-2.54) , D. S.H.−1) followed by L3 treatment (274.64 Kgha−1), whereas in field experiment set 2, available N was maximum in P2 treatment (242.66 Kgha−1) followed by CNS (241.65 Kgha−1). Soil available P also varies with the treatments, and in the field experiment set 1, maximum P was found in CNS-treated soil (24.2 Kgha−1) followed by L3-treated soil (22.6 Kgha−1) and in field experiment set 2 too, a similar pattern was observed. Available K content follows the pattern of available P content. The pH and EC of various treatment soils are mentioned in The availability of soil OC, N, P, and K was enhanced in soils treated with PSB in both field experiments. In the field experiment set 1, the highest OC was found in CNS (0.88%)-treated plots followed by L3 (0.84%)-treated plots. However, in field experiment set 2, the highest OC was observed in L3 (0.78%)-treated plots followed by CNS (0.76%)-treated plots. Available N in field experiment set 1 was highest in CNS treatment (280.44 KghaIn the field experiment set 1 (PSB + RDF), maximum %N, %P, and %K content was found in CNS-treated wheat plants in which values were 0.35, 0.3, and 1.84, respectively, which were on the higher side of compared to uninoculated control (C) in which %N, %P, and %K were found to be 0.26, 0.18, and 1.66, respectively . In fiel−1, respectively, which were on a higher side as compared to uninoculated control (2.12 mgg−1). For Chl B, the maximum response was observed in T4 (0.56 mgg−1)-treated wheat plants followed by P2 (0.35 mgg−1), T3 (0.28 mgg−1), L3 (0.18 mgg−1), and CNS (0.14 mgg−1) which are quite significant from uninoculated control (0.08 mgg−1). In the case of TC and carotenoid, the maximum response was observed in CNS-treated wheat plants which were found to be 2.31 and 0.6 mgg−1, respectively, which were significantly high as compared to uninoculated wheat plants in which TC and carotenoid content was found to be 1.72 and 0.49 mgg−1, respectively. In the field experiment set 2, chlorophyll A was maximum in CNS-treated (1.96 mgg−1) wheat plants, whereas, in L3, T4, P2, and T3, it was found to be 1.61, 1.45, 1.4, and 1.36 mgg−1, respectively. Most of the treatments, except T3, were significantly higher as compared to the uninoculated control (1.46). For Chlorophyll B, TC, and a carotenoid similar pattern was observed in which CNS-treated plants showed a maximum response and the CNS-treated values were 0.69, 2.18, and 0.59 mgg−1, respectively, which were significantly high when compared to their respective control in which the values of Chlorophyll B, TC, and carotenoid were found to be 0.19, 1.43, and 0.64 mgg−1, respectively. Chord analysis of chlorophyll and carotenoid data in both field experiment set 1 and experiment set 2 shows the maximum % increase share by CNS treatment , whereas in P2, T4, L3, and T3, it was found to be 2.76, 2.54, 2.33, and 2.33 mggreatment , B.During ESI-MS analysis, two organic acids, e.g., citric acid and maleic acid, were detected in selected L3 and P2 PSB strains, respectively . The provia plotting the performance of PSB treatments against plant vigor parameters in the component form which further results in selection of L3 and P2 PSB treatment  followed by L3 followed by P2 PSB treatments. These responses have been interpreted reatment .−1h−1) and P2 (78 μg mL−1h−1) was observed when glucose was used as a C source. P solubilization by two strains L3 and P2 in the following sugars is as follows: fructose (L3-74 and P2-72 g mL−1h−1), lactose (L3-62 and P2-64 g mL−1h−1), and sucrose (L3-68 and P2-64 g mL−1h−1), respectively.Maximum inorganic P solubilization in two PSB strains L3 . Fahsi et al. (Pseudomonas lini strain KM349410 recovered from plant rhizosphere (Jujube) solubilized 69 μgmL−1 of inorganic P. The two strains L3 and P2 solubilized P at temperatures <20°C categorizing them as CAMs or psychrotrophic.As phosphorous (P) is one of the major limiting factors in agriculture crop production, P solubilization n et al.  reportedg et al.  reportedi et al.  reportedPseudomonas strain P2 decreased to 4.2. A previous study reported a drop in pH (4.38) during P solubilization by a Bacillus strain   and when used alone showed significant response as compared to uninoculated control, and these results were supported by the study where inoculation of Pseudomonas sp. MS16 resulted in increased wheat biomass and yield  in the medium increases, and this could be the main reason for the pH drop. The pH in analysis , and theanalysis  and citranalysis . The prog et al. . Similarg et al. . A PSB ig et al. , where t−1h−1) and AcP (72.42 μgmL−1h−1) activity was found in CNS-treated soil which is significantly higher than in control where AP and AcP activity was 58 and 64.62 μgmL−1h−1, respectively. The observations were for field experiment set 2 (PSB – RDF). An increase in AP and AcP represents phosphatase activity and, thus, indicates the higher mobilization of free P in soil for utilization by plants. These results were also consistent with an earlier study where greater wheat biomass was accompanied by an increase in AP soil enzyme activity  post-inoculation. AP and AcP activities represent the conversion of organic bound P to the most available inorganic form to plants. In the field experiment set 1, maximum AP (66.64 μgmLPseudomonas), Pikovskaya agar (for positive phosphate-solubilizing bacteria), and Ashbyii (for N fixer) were used. The maximum α-diversity was found in PCA media and α-diversity among the treatment changes with soil status in both field experiment set 1 (PSB + RDF) and field experiment set 2 (PSB – RDF) as analyzed during S.H.E analysis , King's B , and carotenoid content was 2.17, 0.08, 1.72, and 0.49, respectively. This may be due to the greater mobilization of soil P in wheat plants which later, in turn, promotes N content in plants  and field experiment set 2 (PSB – RDF), there was a substantial increase in chlorophyll a, chlorophyll b, total chlorophyll (TC), and carotenoid content in the PSB-treated wheat plants, the and maximum was observed in CNS treatments . In the n et al. , in whicn et al. , a studyPotential plant growth-promoting rhizobacteria including PSM play a significant role in P solubilization -NAIMCC-B-03170 and Pseudomonas aeruginosa (P2)-NAIMCC-B-03171, respectively.Phosphate-solubilizing bacteria strains L3 and P2 have been submitted to the National Bureau of Agriculturally Important Microorganisms (NBAIM) culture collection with the following accession number Dalbergia sissoo Roxb.) and their applications in winter wheat crops for growth and development under field conditions. The present study investigated the P solubilization potential of four PSB strains  in vivo first followed by their application in wheat crops under field conditions. The response of all the PSB treatments in terms of plant agronomic and plant vigor parameters was higher as compared to the uninoculated control. PSB inoculation also promotes soil health and mitigates soil P into wheat plants via promoting NPK content in wheat seedlings, and later, two PSB strains (L3 and P2) and consortia were selected via exhaustive statistical analysis and used for further studies. Low-temperature growth and P solubilization of potential L3 and P2 make them suitable candidates for developing psychrotroph-based biofertilizers that can be used for winter crops. RSM plots also suggest the optimized P solubilization at a temperature <20°C for both L3 and P2 PSB strains.To the best knowledge of our case study, this is the first report of PSB isolated from an agroforestry ecosystem (The original contributions presented in the study are included in the article/HD: writing and original draft preparation. MS: conceptualization, methodology, and supervision. VJ, VS, and LT: editing and reviewing of manuscript. GT: inputs for framing of manuscript. KP: reviewing of manuscript. NB and AK: validation of data. TS: reviewing of manuscript. All authors contributed to the article and approved the submitted version.


Paclitaxel frequently induces peripheral neuropathy and myelosuppression during cancer treatment. According to the National Health Insurance Research Database of Taiwan, traditional Chinese medicine doctors widely use Xiang Sha Liu Jun Zi Tang (XSLJZT) to treat breast cancer patients who have received paclitaxel. We explored the combined therapeutic effects of XSLZJT with paclitaxel. XSLJZT did not exhibit significant cytotoxic effects on P388-D1 cells; however, the combination of XSLJZT (100 and 500 mg/kg) with paclitaxel prolonged the survival rate in P388–D1 tumor-bearing mice compared to paclitaxel-only. In addition, XSLJZT was found to enhance white blood cells (WBC) counts and promote leukocyte rebound in paclitaxel-induced leukopenia in mice. XSLJZT also reduced paclitaxel-induced mechanical pain and inhibited c-Fos protein expression in the L4-6 spinal cords of Wistar rats. Moreover, paclitaxel-induced shortening of the nerve fibers of dorsal root ganglion cells was ameliorated by pre-treatment with XSLJZT. Therefore, we suggest that XSLJZT could be used as an adjunct for cancer patients, as the formula could decrease paclitaxel-induced neuropathy and myelosuppression. Breast cancer is the most common cancer in women. Paclitaxel, a taxane agent, is a widely used chemotherapeutic drug to treat breast cancer. Paclitaxel is a microtubule inhibitor that can inhibit mitosis in cancer cells. It is used in hormone-negative but HER-2 positive, hormone-negative, and HER-2 negative (triple negative) breast cancer patients . PancytoIn Taiwan, the incidence of breast cancer increased by 1.8-fold between 1997 and 2013 . Breast Panax ginseng C. A. Meyer (ginseng), Atractylodes macrocephala Koidz (atractylodes), Wolfiporia extensa (Peck) Ginns (hoelen), Glycyrrhiza uralensis Fisch. ex DC. (licorice), Citrus × aurantium f. Deliciosa (Ten.) M. Hiroe (citrus), Pinellia ternata (Thunb.) Makino (pinellia) and Zingiber officinale Roscoe (ginger). XSLJZT includes two more herbs: Wurfbainia longiligularis. (T.L.Wu). Skornick. and A.D.Poulsen (cardamom), and Dolomiaea souliei (Franch.) C. Shih (saussurea). Preclinical studies indicate that LJZT attenuates cisplatin-induced neurotoxicity , fetal bovine serum, and penicillin/streptomycin, were purchased from GIBCO . Isoflurane was procured from Aesica Queenborough Limited (UK). The anti-c-Fos antibody was purchased from Santa Cruz Biotechnology .Panax ginseng C. A. Meyer, Atractylodes macrocephala Koidz, Wolfiporia extensa (Peck) Ginns, Glycyrrhiza uralensis Fisch. ex DC., Citrus × aurantium f. Deliciosa (Ten.) M. Hiroe, Pinellia ternata (Thunb.) Makino, Dolomiaea souliei (Franch.) C. Shih, Wurfbainia longiligularis. (T.L.Wu) Skornick. and A.D.Poulsen and Zingiber officinale Roscoe were purchased from Sun Tan Pharmaceutical . The medicinal materials were authenticated by a non-profit organization, the Brion Research Institute of Taiwan . Its daily dosage was shown in The prescription of XSLZJT was based on the unified formula announced by the Committee on Chinese Medicine and Pharmacy of the Department of Health . An Agilent 6,470 triple-quadrupole mass spectrometer  equipped with an Agilent Jet Stream electrospray ionization (ESI) source and integrated into the UPLC system Agilent 1,290 Infinity II LC system   was used in this study. The mass spectrometer ion source settings were as follows: Gas Temp, 300 (°C); Gas Flow, 5 (L/min); Nebulizer, 45 (psi); Sheath Gas Heater, 250 (°C); Sheath Gas Flow, 11 (L/min); Capillary ESI+, 4,000 (V); ESI-, 3,500 (V); VCharging, 500 (V). UP-HPLC profile of XSLZJT was analyzed with a mobile-phase gradient elution condition: Water was used for mobile-phase A, and methanol containing 0.1% FA for mobile-phase B. The elution gradient was maintained as follows: 0–20 min, 100% A to 80% A; 20–25 min, 80% A; 25.01–40 min, 80% A to 70% A; 40.01–50 min, 70% A to 60% A; 50.01–60 min, 60% A to 50% A; 60.01–60.10 min, 60% A to 0% A; 60.10–63 min, 0% A. The sample was analyzed using a UV/Vis spectrophotometer at 280 nm. Multiple reaction monitoring (MRM) mode was used during mass spectrometry to detect hesperidin, atractylenoide III, and glycyrrhizic acid. The ion pairs were ESI- m/z 609.3→301.1 , ESI+ 249.1→231.1 , ESI- 821.1→351. .2O. The sample solution was mixed with Folin–Ciocalteu reagent and a 7.5% aqueous Na2CO3 solution. After 5 min incubation at 50°C, the absorbance was measured at 600 nm against water using an enzyme-linked immunosorbent assay (ELISA) reader. The total phenol content was expressed as gallic acid equivalents (mg GA/g sample) using a calibration curve prepared from gallic acid standard concentrations (15.625–250 μg/mL) and was fitted with the following equation: y = 0.00494x − 0.105 (r2 = 0.991).The total phenol content was determined using the Folin-Ciocalteau method . The XSLr2 = 0.997).The total polysaccharide content was determined using the phenol-sulfuric method . The XSLCDF1 mice , BALB/c-nu mice 20 ± 5 g), and Wistar Rats 200 ± 20 g) were purchased from BioLASCO Taiwan Co., Ltd. , housed in a controlled environment at 21°C with sufficient food and water, and kept in an alternating 12 h dark and light cycle. The animal experiments were approved by the Ethical Regulations on Animal Research of Taipei Medical University .2 atmosphere. P388-D1 cells (105 cells/well) were seeded in 24-well plates and co-treated with or without paclitaxel, and the test samples were incubated for 24 h. Cell proliferative activity was detected using a 3--2,5-diphenyltetrazolium bromide (MTT) assay.P388-D1 cells of a mouse macrophage lymphoma cell line were cultured in DMEM with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a 5% CO6 cells/mouse) were intraperitoneally (i.p.) transplanted into 5-week-old female CDF1 mice. The animals were allowed to rest for 24 h before treatment began. The control group was administered distilled water for the entire process. The XSLJZT group was orally administered XSLJZT (500 mg/kg/day) during the entire process. The paclitaxel group (20 mg/kg) received paclitaxel intraperitoneally on days 1, 3, 5, 7, and 9. Paclitaxel (20 mg/kg) and XSLJZT co-administered (100 or 500 mg/kg) group was performed in the order of i.p administration of paclitaxel  and immediately oral administration of XSLJZT (the whole process).P388-D1 cells  calculated according to the following equation: %ILS = [(T/C)–1] × 100%, where T and C respectively represent the mean survival times (days) of the treated and vehicle control groups. The body weight (BW) of each CD2F1 mouse was determined daily on an animal scale. Data are presented as mean ± standard deviation (SD). The Student's t-test was used to compare survival times (days) between the test and blank groups.i.p. Administration of paclitaxel 2 mg/kg once on days 8, 10, 12, and 14, respectively. On day 15, each group was subjected to the Von Frey test for mechanical allodynia. In the post-treatment groups, after i.p. Administration of paclitaxel 2 mg/kg four times, rats were orally administered XSLZJT once a day for 9 days beginning on day 15. On day 23, each group was examined using the Von Frey test for mechanical allodynia and anesthetized with isoflurane to obtain the L4-6 spinal cords.We also designed pre-treatment and post-treatment groups of XSLZJT in the hyperalgesia animal model . Each gri.p.) at 10 and 6.8 mg/kg doses once on days 8 and 14, respectively. In the post-treatment groups, BALB/c mice were orally administered XSLZJT once daily for 9 days after i.p. Paclitaxel administration on day 8. BALB/c mice in the blank group were intraperitoneally injected with normal saline, and sterile water was administered orally. In the induced group, i.p. Paclitaxel was administered, and sterile water was administered orally. Whole blood was obtained from the eyehole vein of BALB/c mice on days 10 and 16. White blood cell (WBC) levels of the whole blood were analyzed using an automatic multi-parameter blood cell counter (Sysmex KX-21 N). The WBC levels of control and XSLJZT compared with the blank group were calculated based on the following equation: Control or XSLJZT/blank × 100%. Data are presented as mean ± SD.XSLZJT pre-treatment and post-treatment were designed for paclitaxel-induced leukopenia in a BALB/c mouse model . BALB/c The Von Frey test was performed as described previously  and asseL4-6 spinal cords fixed in 10% (v/v) neutral buffered formalin for 24 h, paraffin-embedded, and cut into 5 μm thicknesses for histopathological assessment . c-Fos i2.Six-week-old male Wistar Rats were anesthetized with isoflurane, the spinal cord was removed, the DRG was harvested, and was cleaned under a stereoscopic microscope. DRG were dissociated into single cells using a two-step enzyme treatment. First, chopped DRG was treated with 0.125% collagenase type I at 37°C for 90 min with gentle shaking. The cell pellet was collected by centrifugation at 1,500 × g for 5 min. Second, the cell pellet was incubated with 0.25% trypsin-EDTA solution at 37°C for 30 min and triturated with fire-polished Pasteur pipettes with decreasing tip diameter. Next, the cells were plated on poly-D-lysine (PDL)-coated 8-well chamber slides and 24-well microtiter culture plates incubated in Ham’s F12 medium containing 10% heat-inactivated fetal bovine serum, followed by 1% penicillin/streptomycin for up to 5 days. The cultures were maintained at 37°C in a humidified atmosphere containing 5% COA stock solution of XSLJZT (10 mg/mL) was prepared by dissolving the test samples in ddH2O and storing the mixture at 4°C until further use. On the 6th day of culture, test samples at the appropriate concentrations were added to the culture plates for 24 h without changing the medium in serum-free medium with NGF (10 ng/mL). The number of surviving cells in 24-well microtiter culture plates was counted using the tetrazolium (MTT) assay . The cytThe DRG cells, treated with or without test samples on 8-well chamber slides, were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min at room temperature permeabilized with PBS containing 0.2% Triton X-100 and blocked with goat serum and BSA. Next, the cells were immunostained with a polyclonal antibody against peripherin  as a sensory nerve marker. Alexa Fluor 488-conjugated goat anti-rabbit IgG dilution (1:200) was used as the secondary antibody. The slides were mounted using Mount Quick Mounting Medium. Fluorescent images (ten images per treatment) were captured using a fluorescence microscope . The length of neurites positive for peripherin was measured using ImageJ software . At least 15 cells from triplicate wells per group were analyzed for each experiment.p < 0.05.Results are presented as mean ± standard deviation (SD). Data were analyzed using the SPSS software . Group differences were statistically assessed by one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test to compare means. Statistical significance was set at In this experiment, we used UP-HPLC fingerprint profile to confirm the possible components in XSLJZT. The results show that hesperidin, atractylenoide III, and glycyrrhizic acid as the major compounds in XSLJZT . Further50) of 11.34 ± 0.33 µM. XSLJZT exhibited an inhibitory effect on P388-D1 cells at a 5 mg/mL concentration with an IC50 value of 2.98 ± 0.81 mg/mL. The P388-D1 cells were treated with paclitaxel and XSLJZT. At a high dose (5 mg/mL), XSLJZT significantly enhanced toxicity toward P388-D1 cells. . It significantly inhibited P388-D1 cells with a 50% inhibitory concentration  significantly improved the survival rate compared to the control group by 44.9% and 55.4%, respectively. However, the combination of XSLJZT with paclitaxel increased by 11.1% compared to the group receiving paclitaxel. These results suggest that XSLJZT does not influence the in vivo antitumor effect of paclitaxel.The antitumor effects of paclitaxel alone and paclitaxel combined with XSLJZT were evaluated using P388-D1 cells in an We studied the preventive and treatment effects of XSLJZT on paclitaxel-induced leukopenia in mice. The study procedure is illustrated in i.p. administration of 2 mg/kg paclitaxel. Subsequently, both groups received paclitaxel once every 2 days up to 4 times. Mechanical allodynia tests were performed on days 15 and 23. On day 23, the animals were sacrificed to harvest their L4-L6 vertebra for pathological examination. The results showed that, on day 15, pain sensitivity was significantly elevated in the control group , XSLJZT inhibited P388-D1 cell proliferation. Although XSLJZT inhibited P388-D1 cells in vitro, it did not exhibit antitumor effects in P388-D1 CD2F1 tumor-bearing mice. Paclitaxel demonstrated inhibition of P388-D1 cells both in vitro and in vivo. In this study, co-administration with different dosages of XSLJZT slightly improved the survival rate of P388-D1 CD2F tumor-bearing mice, with an 11.1% increase in the lifespan compared to the group receiving paclitaxel alone. It was concluded that XSLJZT did not exert cytotoxic effects against P388-D1 cells in vivo without influencing the inhibitory effects of paclitaxel in P388-D1 CD2F tumor-bearing mice. In a meta-analysis, it was observed that patients receiving chemotherapy with a combination of herbal medicines had a higher survival rate than those receiving chemotherapy only.Seven out of the nine constituents of XSLJST isolated from t cancer . Two of t cancer . The cytPaclitaxel causes leukopenia in approximately 78%–100% of patients. Chemotherapy-associated myelosuppression commonly occurs after the first course of treatment; however, evidence also suggests that leukopenia may be associated with longer progression-free survival . GranuloChemotherapy-induced peripheral neuropathy (CIPN) is another frequently occurring condition that causes cancer patients to undergo suffering from the use of lifesaving therapies. Clinically, CINP is a symmetrically distributed sensory axonal neuropathy that starts in the fingers and toes and progresses toward the body. Paclitaxel-induced CINP is highly prevalent, dose-dependent, and usually treated by discontinuing the treatment. The mechanisms involved in this type of CINP include taxane-induced microtubule disruption that leads to Wallerian degeneration and neuroinflammation, resulting in atrophy of the DRG, hyperexcitability of peripheral neurons, axon degeneration, and secondary demyelination of peripheral nerves . c-Jun aIn addition to myelosuppression and neuropathy, chemotherapy induces many unexpected side effects, such as functional dyspepsia and diarrhea . These gIn conclusion, our study supports the finding that XSLZJT does not exert cytotoxic effects in P388-D1 CD2F tumor-bearing mice and does not significantly interfere with the therapeutic effects of paclitaxel. XSLJZT reduces paclitaxel-induced allodynia and immunosuppression. Based on our results, we suggest that XSLJZT could be prescribed after paclitaxel to reduce neurotoxic and leukopenia side effects and improve the patient’s quality of life .


Millettia dielsiana against the target proteins PDE4  showed binding affinity ranges from −5.81 to −11.56, −5.27 to −13.01, and −5.80 to −12.12 kcal mol−1, respectively, with median values of −8.83, −8.84, and −8.645 kcal mol−1, respectively. Among these compounds, Millesianin F was identified as the most promising PDE4A inhibitor due to its strongest binding affinity with the target protein PDE4A. . This was followed by the compound 5,7,4′-trihydroxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (D50) with the binding affinity value of −11.35 kcal mol−1. For the target protein PDE4B, compound D50 exhibited the strongest binding affinity value of −13.01 kcal mol−1, while showing poorer inhibition ability for PDE4D. The 100 ns MD simulation examination , Root-Mean-Square Deviation (RMSD), Root-Mean-Square Fluctuation (RMSF), and hydrogen bonding) was carried out to examine the overall stability and binding efficiency of the protein–ligand complex between compounds -β-d-glucopyranoside, 7-hydroxy-4′,8-dimethoxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside, Odoratin-7-O-β-d-glucopyranoside, and 5,7,4′-trihydroxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside) and PDE4  subtype proteins. Compound D50 has shown strong anti-inflammatory activity, as evidenced by experimental results. It effectively inhibits PDE4B and PDE4D, with IC50 values of 6.56 ± 0.7 µM and 11.74 ± 1.3 µM, respectively. Additionally, it reduces NO production, with an IC50 value of 5.40 ± 0.9 µM. Based on these findings, it is promising and considered a potential novel anti-inflammatory drug for future development.The results of in silico screening of the 50 isolated compounds from  A vast array of PDE4 inhibitors have been evaluated in clinical trials for various inflammatory conditions. Many drugs have considerable efficacy but also have adverse effects such as nausea and emesis, which limit their dosing and subsequently their immunomodulatory activity. Thus, developing PDE4 inhibitors that retain anti-inflammatory effects while limiting side effects has been a significant focus of pharmaceutical research for treating inflammatory diseases  and saved as CDX format files. The 2D sketched compounds were converted to 3D structures by 3DView 2021.1.2 software, followed by energy minimization then prepared for docking. For parameter preparation, the grid box was centered on the active site pocket and set to an exhaustiveness of 400. As a result of these processes, the predicted docking pose for each ligand was visualized by Discovery Studio Visualizer v2021 software   ,30,31.+ and Cl- were introduced into the system at constant pressure (1.0 bar) and temperature (300 K) to balance the overall charge. The coordinates of the ligands were prepared to transform them into topologies appropriate for GROMACS using AmberTools22 sample − [XTB]LPS)/([XTB]ĐC − [XTB]LPS) × 100Cell RAW264.7  was cultured for 48 h in DMEM (Dulbecco’s Modified Eagle Medium) medium at 37 °C, 5% COTB] is the average NO concentration calculated based on the standard NaNO2.In which [X2 incubator at 37 °C. Formazan crystalline metabolites are dissolved in dimethyl sulfoxide  and measured optical density at λ = 540/720 nm on Infinite F50 device  [The remaining cell portion after being used to evaluate in vitro activity was supplemented with MTT solution (0.5 mg/mL in PBS), incubated for 4 h in a 5% COzerland) ,44.anti-inflammatory activity in general. Further studies can focus on finding new anti-inflammatory agents from isoflavonoids. The chemical composition of Millettia dielsiana, which is rich in these active compounds, has contributed to clarifying the folk experience of treating arthritis and protecting the liver, and rheumatism.Isoflavonoids, D38, 50, 45, 47, D41, D39, and D42 interacted well with the active enzyme pocket of PDE4 . This has suggested good activity for the PDE4 enzyme in particular and Millettia dielsiana showed a predicted LD50 of 5000 mg/kg and were almost safe by toxicity prediction in silico methods. Especially among them, compound D50 has no toxicity against any of the studied targets. Compound D41 was predicted to be cytotoxic, and might interact with AhR. And compound D38 demonstrated activity in interacting with p53. Determining the toxicity of compounds is necessary, an important step in drug design, and has great meaning in saving time, finances, and morality.All selected compounds from O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (D50) may be suggested as a potent candidate for anti-inflammatory drug. This study primarily focuses on the in silico screening and in vitro experimental of anti-inflammatory activity. Therefore, research on in vivo activity, acute toxicity, and the correlation between them is a promising future research direction.Compound 5,7,4′-trihydroxyisoflavone 7-


Transcriptomic and functional analysis demonstrated metformin-mediated alterations to energy metabolism, mitochondrial function, cell cycle distribution and progression, cell death and antioxidant levels in colorectal cancer cells. Using ex vivo models, metformin treatment significantly inhibited oxidative phosphorylation and glycolysis in treatment naïve rectal cancer biopsies, without affecting the real-time metabolic profile of non-cancer rectal tissue. Importantly, metformin treatment differentially altered the protein secretome of rectal cancer tissue when compared to non-cancer rectal tissue. Together these data highlight the potential utility of metformin as an anti-metabolic radiosensitiser in rectal cancer.Resistance to neoadjuvant chemoradiation therapy, is a major challenge in the management of rectal cancer. Increasing evidence supports a role for altered energy metabolism in the resistance of tumours to anti-cancer therapy, suggesting that targeting tumour metabolism may have potential as a novel therapeutic strategy to boost treatment response. In this study, the impact of metformin on the radiosensitivity of colorectal cancer cells, and the potential mechanisms of action of metformin-mediated radiosensitisation were investigated. Metformin treatment was demonstrated to significantly radiosensitise both radiosensitive and radioresistant colorectal cancer cells  An estiity rate , who oftity rate , 8.The current standard of care for locally-advanced rectal cancer is neoadjuvant chemoradiation therapy (neoCRT), followed by surgical resection. Patients typically receive long-course radiation therapy, combined with 5-fluorouracil (5-FU) based chemotherapy prior to total mesorectal excision (TME) . Increasin vitro and ex vivo models of oesophageal adenocarcinoma . SW837 cells were maintained in Leibovitz’s (L-15) culture media , supplemented with penicillin-streptomycin (1%) (v/v) (Lonza), foetal bovine serum (FBS) (10%) (v/v) , and L-Glutamine (Lonza) (1%) (v/v) (complete medium), in non-vented flasks. HCT116 colon cancer cells were maintained in Roswell Park Memorial Institute (RPMI)-1460 medium (Gibco), supplemented with penicillin-streptomycin (1%) (v/v), and FBS (10%) (v/v) (complete medium) (complete RPMI (cRPMI)), in vented flasks. Cell lines were cultured at 37°C, in 5% CO2.22O) for 30 min at RT°. Cells were gently washed with 50 µL water and allowed to air dry overnight at RT°. The crystal violet dye was then eluted via the addition of 50 µL 1% Triton-X100 (in PBS) on an orbital shaker for ~ 1 h, or until the dye fully eluted, at RT°. The absorbance was read at 595 nm on a VersaMax microplate reader .SW837 cells were fixed via the addition of 50 µL 1% glutaraldehyde [(v/v) in phosphate buffered saline (PBS)], per well) for 15 min, at room temperature (RT°). HCT116 cells were fixed with cold 4% paraformaldehyde (PFA) (v/v) for 10 min, at RT°. The fixative was removed and cells were washed with 50 µL PBS. Cells were stained with 0.1% crystal violet (w/v)   or vehicle control (H2O) diluted in cRPMI. Following 24 h, treatment was removed and cells were washed with unbuffered Seahorse XF Base DMEM (Agilent) (supplemented with 10 mM glucose (Sigma), 10 mM sodium pyruvate (Sigma) and L-glutamine) and placed in a non-CO2 incubator for 1 h at 37°C. OCR and ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyser (Agilent). Three baseline measurements of OCR and ECAR were taken over 24 min, consisting of 2 repetitions of mix (3 min)/wait (2 min)/measurement (3 min), to establish basal respiration. All OCR/ECAR readings were normalised using the crystal violet assay.Cells were seeded at optimised seeding densities  in a 24-well cell culture XFe24 microplate  and allowed to adhere at 37°C, 5% CO2.4FM), as previously described  or vehicle control (H2O) diluted in cRPMI. At 24 h post treatment, the media was removed and cells were incubated with a fluorescent probe {in PBS with magnesium [PBS (Mg2+)]} for 30 min, in the dark at 37°C. The probe was removed, fresh PBS was added and the fluorescence was immediately read using a FLx800 Fluorescence microplate reader (Mason Technology). Fluorescence values were subsequently normalised to cell number using the crystal violet assay.Three surrogate markers of mitochondrial function, reactive oxygen species (ROS), mitochondrial membrane potential and mitochondrial mass were assessed using a series of fluorescent probes , Rhodamine-123 and MitoTracker Greenescribed . Briefly2.5All irradiations were performed using an X-Strahl cabinet X-ray irradiator (RS225) (X-Strahl) at a dose rate of 1.74 Gy/min.2.62/95% humidified air overnight. Following 24 h incubation, medium was removed to waste, and cells were treated with metformin  or vehicle control (H2O) diluted in cRPMI. After 24 h, cells were exposed to X-ray radiation at 1.8 Gy or were mock-irradiated. For 5-FU treatments, cells were treated with 5-FU (15 µM) or DMSO control (0.001%) for 6 h prior to irradiation. At 24 h post-irradiation, treatment medium was removed and replenished with 1.5 mL/well cRPMI. Cells were incubated at 37°C in 5% CO2/95% humidified air for 7-14 days, until colonies formed but did not merge.HCT116 and SW837 cells in the exponential growth phase were harvested, seeded in cRPMI into 6-well plates at optimised cell densities  and were allowed to adhere at 37°C in 5% CO2.7For SW837 cells, a volume of 500 µL fixing/staining solution (0.05% (w/v) crystal violet (Sigma), 25% (v/v) Methanol (Sigma) in PBS) was added to each well and incubated for 30 min, at RT°. For HCT116 cells, a volume of 500 µL 4% PFA (4°C)  was added to the well and incubated for 10 min at RT°. Colonies were stained with crystal violet solution (0.05% (w/v)) for 30 min at RT°. The stain was removed to waste, and the wells gently washed with water. Plates were then left to air-dry overnight. Colonies were counted using a PC-software operated colony counter . Plating efficiencies (PEs), based on control colony counts, were calculated using the following formula: PE = no. colonies/no. cells seeded. The surviving fraction (SF) was calculated using the following formula: SF = No. colonies formed after treatment/(No. cells seeded x PE) as previously described .2.82/95% humidified air. At 24 h, medium was removed and cells were treated with metformin (10 mM) or vehicle control (H2O) diluted in cRPMI. Twenty-four hours later, cells were mock-irradiated or exposed to 1.8 Gy X-ray radiation. At, 6 h, 10 h, or 24 h post-irradiation, cells were collected into 5 mL flow tubes, and stained with propidium iodide (PI). Samples were acquired, with a minimum of 10,000 events collected, excluding doublets, using the FACSCanto II flow cytometer (BD Biosciences). PI was measured on the PerCP-Cy5 channel. Data were analysed using FlowJo™ Version 10.6.2.Cells were seeded into 12-well plates at optimised densities  in cRPMI and allowed to adhere overnight at 37°C in 5% CO2.92/95% humidified air. At 24 h, medium was removed and cells were treated with metformin (10 mM) or vehicle control (H2O) diluted in cRPMI. After 24 h, cells were irradiated with 1.8 Gy X-ray radiation, while controls were mock-irradiated. At 24 h post radiation, supernatants and cells were collected into 5 mL flow tubes. Cells were stained with Annexin-V-FITC for 15 min at 4°C. Cells were then stained with PI prior to acquisition of 40,000 cells per tube, excluding doublets using the FACSCanto II flow cytometer. Annexin-V-FITC was measured on the FITC channel, while PI was measured on the PerCP-Cy5 channel. Data were analysed using FlowJo™ Version 10.6.2.Cells were seeded in cRPMI into 12-well plates at optimised seeding densities  and allowed to adhere overnight at 37°C in 5% CO2.102O) diluted in cRPMI and incubated at 37°C in 5% CO2/95% humidified air. After 24 h treatment, media was removed to waste and GSH/GSSG levels measured using the GSH/GSSG-Glo™ luminescent assay (Promega), according to the manufacturer’s instructions. Luminescence was measured at 1 s integration time using the Explorer Luminometer (Promega). An identical plate of cells was set up and treated as with the experimental plate and was used for normalisation of assay results by crystal violet assay.Cells were seeded in cRPMI in white-walled 96-well plates  at a density of 15,000 cells/well. At 24 h post seeding, medium was removed to waste, and cells were treated with 100 µL of metformin (10 mM) or vehicle control (H2.112O) diluted in cRPMI for 24 h. Total RNA was isolated from cells using the miRNeasy® Mini Kit , according to manufacturer instructions. RNA was quantified using a Nanodrop 1000 spectrophotometer version 3.1 .SW837 cells were treated with metformin (10 mM) or vehicle control , according to the manufacturer instructions, using a starting volume of 50 ng RNA. An equal molar amount of the purified library was pooled for sequencing, with a loading concentration of 320 pM loaded onto the NovaSeq flowcell. Sequencing was performed using the NovaSeq 6000 , and an SP v1.5 sequencing kit (Illumina) with 1 x 100bp reads, as per manufacturer instructions.2.13Raw files were assessed using the BlueBee™ Bioinformatics platform (Lexogen). Raw reads were trimmed and aligned for automated gene counting. Once gene reads and counts were complete, differential expression analysis was performed using the DESeq2 R script extension within BlueBee software.2.142 Fold Change values were imported to IPA bioinformatics software . Core analysis in IPA was performed, which utilises the Qiagen Knowledge Base, to identify networks and predict specific biological function and pathway involvement in the uploaded experimental transcriptomic dataset. Canonical Pathway Analysis in IPA, utilising the Qiagen Knowledge Base, was utilised to predict involvement and activation or inhibition of specific biological pathways in the experimental dataset. The p-value denotes the significance between the overlap of input experimental data and the Ingenuity Knowledge Base, indicating confidence in pathway involvement. The Z-score refers to software prediction of the activation or inhibition of each affected canonical pathway, with a Z-score ≥ 2, or ≤ -2 indicating significant activation or inhibition of each pathway, respectively.Significantly differentially expressed genes, and corresponding Log2.15Ethical approval for patient sample collection for this study was granted by the Joint St. James’s Hospital/AMNCH ethical review board (Reference 10/11/2011) and the Beacon Hospital Research Ethics Committee (Reference BEA0139). Patients undergoing lower gastrointestinal investigations or endoscopy for rectal cancer diagnosis were recruited between October 2020 and January 2022 from St. James’s Hospital, Dublin and Beacon Hospital, Dublin. Pre-treatment rectal tumour biopsies were obtained from consenting patients at diagnostic endoscopy. Normal (non-cancer) rectal tissue biopsies were obtained during colonoscopy from consenting patients who did not have a cancer diagnosis. Histological confirmation of tumour tissue and non-malignant tissue in biopsies was performed by an experienced pathologist using haematoxylin and eosin staining.2.16Protein was isolated from patient tissue biopsies using an AllPrep DNA/RNA/Protein Mini Kit (Qiagen), according to manufacturers’ instructions. To quantify the protein content of patient biopsies, the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was utilised, according to the manufacturer’s instructions.2.172/95% humidified air for 30 min to equilibrate. Three basal measurements of OCR and ECAR were measured over 24 min of three repeats of mix (3 min)/wait (2 min)/measurement (3 min) using the Seahorse XFe24 analyser. The biopsies were treated with metformin (10 mM) or H2O control in complete M199 medium (1 mL volume). Biopsies were cultured for 24 h at 37°C in 5% CO2/95% humidified air, and basal measurements of OCR and ECAR measured over 24 min as described. Biopsies and matching tumour conditioned media (TCM) or non-cancer conditioned media (NCM) were collected, snap-frozen in liquid nitrogen, and stored at -80°C until required. The effect of treatment was assessed as the change in OCR/ECAR measurements following vehicle/metformin treatment, when compared to each corresponding baseline measurement. Metabolic rates were normalised to protein content using the Pierce BCA assay as per the manufacturers’ instructions.Two biopsies per patient were collected at colonoscopy, placed into an individual well of an XF24 Islet Capture Microplate (Agilent Technologies) and secured into place by islet capture screens. A volume of 1 mL complete M199 (Gibco)  was placed in each well. The plate was placed at 37°C, in 5% CO2.18To assess angiogenic, vascular injury, pro-inflammatory, cytokine and chemokine secretions a 54-plex ELISA kit separated across 7 plates was used . The multiplex kit was used to quantify the secretions of CRP, Eotaxin, Eotaxin-3, FGF(basic), Flt-1, GM-CSF, ICAM-1, IFN-γ, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-1RA, IL-1α, IL-1β, IL-2, IL-21, IL-22, IL-23, IL-27, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IL-9, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, PlGF, SAA, TARC, Tie-2, TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C and VEGF-D. All assays were run as per manufacturer’s recommendation, an overnight supernatant incubation protocol was used for all assays except Angiogenesis Panel 1 and Vascular Injury Panel 2, which were run according to the same day protocol. TCM and NCM were run undiluted on all assays except Vascular Injury Panel 2, where a one-in-four dilution was used. Secretion data for all factors was normalised to cell lysate protein content by using a BCA assay.2.19post-hoc Tukey’s multiple comparisons testing or t-testing, depending on the experimental set up, as described in figure legends. For transcriptomic data analysis, BlueBee, DESeq2 R extension and IPA software were utilised for statistical analysis. DESeq2 utilised Wald testing, while IPA utilised Fisher’s Exact Test, as stated in figure/table legends. Analysis on patient samples used Mann-Whitney U or Wilcoxon signed rank test, as appropriate. Results were considered significant where probability (p) ≤ 0.05.All statistical analysis and graphing were performed using Graphpad Prism v9 software. Data is presented as mean ± standard error of the mean (SEM) throughout. Statistical comparisons were carried out using analysis of variance (ANOVA) testing, 33.1in vitro, the metabolic phenotypes of HCT116 colon cancer and SW837 rectal cancer cells treated with metformin were assessed by Seahorse profiling. This permits the measurement of the two major metabolic pathways, oxidative phosphorylation, represented by oxygen consumption rate (OCR), and glycolysis, represented by extracellular acidification rate (ECAR) in live cells in real-time.To investigate the metabolic modulating effects of metformin treatment on CRC cells in vitro.Metformin treatment (2.5 mM and 10 mM) significantly reduced OCR in HCT116 cells, when compared to vehicle control  significantly sensitised radiosensitive HCT116 cells to 1.8 Gy radiation, when compared to vehicle control  underlying this metformin-mediated radiosensitisation were investigated. Basal cell cycle distribution has been implicated in the radio response, with cells in the S phase and G0/G1 phases being more radioresistant, and cells in the G2/M phase being the most sensitive to radiation exposure .Metformin treatment significantly reduced the proportion of radiosensitive HCT116 cells in the G0/G1 phase at 48 h post treatment, when compared to vehicle control  or HIn radiosensitive HCT116 cells, the addition of metformin to 1.8 Gy X-ray radiation significantly increased the proportion of G0/G1 cells at 6 h post irradiation, but by 24 h post irradiation, the proportion of G0/G1 cells were significantly reduced in cells treated with metformin and 1.8 Gy, when compared to irradiated vehicle control  significantly reduced the number of live HCT116 and SW837 cells, when compared to vehicle control .p-value (p-adj) < 0.05  were collected from consenting patients undergoing diagnostic endoscopy, a baseline measurement of metabolic rate was recorded, biopsies were treated for 24 h with metformin (10 mM) or H2O vehicle control and metabolism was assessed at 24 h post treatment. The patient cohort characteristics are outlined in Having demonstrated metabolic modulatory effects of metformin in an Metformin significantly inhibited OCR by 47%  taken from non-cancer patients was assessed in real-time using the Seahorse XFe analyser. Whilst significant alterations in OCR and ECAR were demonstrated in normal rectal tissue biopsies treated with H2O vehicle control versus baseline, interestingly, no significant alterations in OCR or ECAR was demonstrated following metformin treatment, when compared to baseline  and normal, non-cancer conditioned media (NCM) samples from biopsies treated with either metformin or HIn TCM samples, 7 proteins were demonstrated to be significantly altered in metformin treated samples, when compared to vehicle control , with enhanced mitochondrial targeting capacity, have been demonstrated to enhance metformin-mediated inhibition of mitochondrial respiration and radiosensitisation in prostate cancer  and Beacon Hospital Research Ethics Committee (Reference BEA0139). The patients/participants provided their written informed consent to participate in this study.Study Design: CB, JO’S, and NL-L; Data curation: CB, RO’B, TN, and ND; Collection of clinical samples: TN, ND, AH, DO’R, RH, PN, RK, BM, PM, CD, MK, and JL; Performed Experiments: CB; Data analysis: CB, FO’C, NL-L, and JO’S; Funding acquisition: NL-L. Manuscript Writing and Revision: CB, JR, JO’S, and NL-L. All authors contributed to the article and approved the submitted version.


These CPs were characterized by using different techniques, including single-crystal X-ray diffraction. The structural studies demonstrated that in 2, there are four Zn(II) centers and both centers are in different coordination environments . Hirshfeld surfaces analysis revealed that different types of intermolecular interactions  are present in the synthesized CPs. We examined the different antibiotics, such as metronidazole (MDZ), nitrofurazone (NFZ), dimetridazole (DTZ), sulfasalazine(SLA), and oxytetracycline (OXY), degradation behaviors of the synthesized CPs, which showed remarkable degradation efficiency. 1 showed photocatalytic behavior toward the NFZ antibiotic in an aqueous media. This study also showed that these catalysts are stable and reusable under mild conditions.The efficient removal of organic contaminants from wastewater is, nowadays, a prominent area of study due to its biological as well as environmental significance. Antibiotics are now found in wastewater because of their high use, which has become a source of aquatic pollution. These antibiotics have dangerous implications for people’s health. Hence, effective pharmaceutical removal from wastewater and contaminated water bodies, especially the removal of antibiotics, is of major interest to global research organizations. This is why it is necessary to investigate this class of toxic material in wastewater discharge. We synthesized three different coordination polymers (CPs) in the presence of various assistant carboxylate linkers, namely, [Zn(Hbtc)(dip)] Various harmful and persistent organic contaminants in water pose a serious threat to ecological security, making water pollution now one of the most serious worldwide environmental problems ,2,3,4. MCoordination polymers (CPs) are porous crystalline resources that have received considerable attention in the last two decades due to their intriguing assemblies with cationic central parts connected by specific multidentate organic ligands ,15,16,172 reduction n (2), and [Zn(dip)]n (3) pyridine = dip, 1,3,5-benzenetricarboxylic acid = H3btc, 1,2-benzenedicarboxylic acid = 1,2-H2bdc, and 1,4-benzendicarboxylic acid = 1,4-bdc), were solvothermally synthesized  and different carboxylate linkers, three Zn-containing frameworks, namely, [Zn(Hbtc)(dip)]thesized . This inThis study examines the use of CPs as catalysts in the photocatalytic degradation of various water pollutants. They exhibit interesting various structures due to the different assistant carboxylates used in the reaction. The degradation of antibiotics, such as metronidazole (MDZ), nitrofurazone (NFZ), dimetridazole (DTZ), sulfasalazine (SLA), and oxytetracycline (OXY), in an aqueous media are investigated in detail.1 revealed that it crystallizes in the triclinic system and P-1 space group  can be described as distorted tetrahedral{ZnN2O2} (1 contains one atom of Zn(II), one Hbtc molecule, and one dip. An intriguing secondary building unit (SBU)-based 3D porous network development is shown by the structural analysis. The dip molecules form the typical 1D helical chain of extended-SBU, while the acid compounds connect these ext-SBUs at the different metal centers to form a 2D structure  centers, four 1,2-bdc ligands, and four dip ligands. Zn2 is coordinated by two monodentate oxygen atoms from two 1,2-bdc ligands and two nitrogen atoms from two dip ligands, having the formation of distorted tetrahedral geometry  ion, one 1,4-bdc ligand, and one dip ligand in the asymmetric unit. The Zn1 ion possesses a distorted tetrahedral structure composed of two nitrogen atoms from two dip ligands  Å)  Å)  and two 13(4) Å) a. The th13(4) Å) d.1−3 were determined in the temperature range of room temperature (RT) to 800 °C. The TG experiment was conducted in a N2 environment on the crystalline samples of 1–3. The temperature was increased at 10 °C/min from 40 to 800 °C  method is useful for identifying organic materials and functional groups in a variety of substances. FTIR spectra were obtained for 1–3 . The speectively . Strong 1–3 were calculated using the diffuse reflectance (DR) UV-vis data. The subsequent values were determined as the cross-site between the x-axis, and the lines were inferred by the straight line position of the absorption boundary. The Eg values obtained were 3.76, 3.82, and 3.93 eV , and reaction time, the kinetic rate constant, k, was calculated [A photocatalytic degradation experiment on various antibiotics was carried out to evaluate the photocatalytic activity of the synthesized compounds under the influence of visible light irradiation. Photocatalysts were first immersed in antibiotic solutions and reacted in the dark for 30 min to reach the equilibrium of adsorption/desorption. Stirring was continued during the photodegradation reaction to maintain the mixture in suspension. At regular intervals, samples were collected and centrifuged to isolate the photocatalyst for analysis. The UV−vis absorption characteristics of antibiotics decreased gradually with time  in the pobserved e, which observed f, ln affected the effectiveness of photodegradation utilizing 1. According to the findings, 76% of NFZ was photodegraded at 10 ppm, while at 20 ppm, NFZ’s photodegradation efficiency increased, and approximately, 87% of the initial NFZ was destroyed after 45 min of irradiation. Then, the percentage of decomposition decreased to 70% when the NFZ concentration increased (to 30 ppm) d.+), 1 mM benzoquinone , or 1 mM tertiary butyl alcohol  [+ was not the major active species either. However, the degradation of antibiotics was significantly inhibited by the addition of BQ and TBA, indicating that O2˙− and ˙OH played a crucial role [To determine the active species for the degradation of antibiotics, trapping experiments were performed using different scavengers, such as by adding 1 mM ammonium oxalate    and exterior surface . The Hirshfeld dnorm surface was used for the description of the intermolecular interactions. Dark-red spots act for effective classical interactions in the Hirshfeld surface analysis, and light-red spots act for non-classical interactions or Van der Waals interactions.Crystal Explorer software is generally applied in Hirshfeld surface analysis, especially in the study of intermolecular interactions and exterior molecular environments. The intermolecular interactions of 1, C···H/H···C and H···H interactions reflect 13.2 and 48.3%, respectively; the amount of which is more than half of the total. The proportions of O ···H/H ···O, C···C, and N···H interactions comprise 30.4%, 5.4%, and 9.1%. The proportion of O···H/H···O interactions is 20.6% of the Hirshfeld surfaces of 1,2-bdc in 2, whereas in 3, it is 11.6%, suggesting that imidazole interacts with the O-atoms of the carboxylate groups. The proportions of O···H/H···O interactions are responsible for the formation of a stronger framework. The percentage of non-classical interactions viz. C···H/H···C, C···C, and H···H are 7.1%, 5%, and 38% in 2 and 8.9, 6.7, and 39.2 in 3. A close investigation of the Hirshfeld surface analysis of these CPs showed that the maximum area of the Hirshfeld surface is comprised of O···H/H···O and H…H types of interactions  spectrum in KBr disc was recorded using Nicolet Impact 750 FTIR between 400 and 4000 cm−1. Thermogravimetric analysis (TGA) was performed under nitrogen atmosphere from room temperature to 900 °C with a heating rate of 10 °C min−1. The Cif files for the structures of CPs 1–3 were deposited with the Cambridge Crystallographic Data Centre (CCDC) as deposition nos. 1–3 and are 2,190,809, 2,190,811, and 2,190,810.All reagents and solvents were analytical-reagent-grade. The powder X-ray diffraction (PXRD) spectra were obtained by using a Bruker D8 diffractometer with monochromatic  radiation with a scanning rate of 6°/min and a step size of 0.02°. The structures were solved by direct methods and refined by full-matrix least-squares on F3)2·6H2O , bip , H3btc , and 6 mL of CH3CN/H2O (v/v = 1:1) was added to 2d NaOH (0.5 mol/L), and the mixture was stirred for 30 min. The mixture was placed in a Teflon-lined reactor and heated at 120 °C for three days. White crystals were obtained after cooling the reaction mixture to room temperature. The crystal was washed with MeOH. The yield was 63% based on Zinc. Elemental anal. calcd. C20H13N5O6Zn: C, 49.46; H, 2.56; N, 14.48; Found: C, 50.01; H, 2.53; N, 14.57.A mixture solution of Zn(NO3)2·6H2O , and 6 mL of CH3CN/H2O (v/v = 1:1) was combined, and the mixture was stirred for 30 min. The mixture was placed in a Teflon-lined reactor and heated at 120 °C for three days. White crystals were obtained after cooling the reaction mixture to room temperature. The crystal was washed with MeOH. The yield was 70% based on Zinc. Elemental anal. calcd. C19H13N5O4Zn: C, 51.78; H, 2.97; N, 15.89 Found: C, 51.99; H, 3.00; N, 15.97.A mixture of bip , 1,2-bdc , Zn(NO3)2·6H2O , and 6 mL of CH3CN/H2O (v/v = 1:1) was combined, and the mixture was stirred for 30 min. The mixture was placed in a Teflon-lined reactor and heated at 120 °C for three days. White crystals were obtained after cooling the reaction mixture to room temperature. The crystal was washed with MeOH. The yield was 66% based on Zinc. Elemental anal. calcd. C38H26N10O8Zn2: C, 51.78; H, 2.97; N, 15.89; Found: C, 51.96; H, 3.01; N, 15.96.A mixture of bip , 1,4-bdc , Zn(NO1–3 (40 mg) were evaluated by the degradation of antibiotic pollutants in the aqueous solution under a UV-400-type photochemical reactor with a 400 W mercury lamp . An aqueous solution of 50 mL 20 mg/L antibiotics was mixed with 0.01 mmol photocatalyst. The suspension containing antibiotics and photocatalysts was magnetically stirred for 30 min in the dark until adsorption–desorption equilibrium was established. A 3.5 mL sample was extracted at 5 min intervals using 3 mL pipettor and centrifuged to remove the residual catalyst for analysis on a UV–visible spectrophotometer at an absorption wavelength applied to monitor the photocatalytic degradation. In addition, a control experiment was also accomplished with the following reaction conditions: (1) without photocatalyst under UV irradiation; (2) with photocatalyst under UV irradiation in the presence of 2 mL tert-butanol (t-BuOH); (3) 2 mL benzoquinone (BQ) was used instead of t-BuOH; (4) 2 mL ammonium oxalate (AO) was used instead of TBA. The degradation efficiency of antibiotics is defined as follows:0 (mg/L) is the initial concentration of dyes, and C (mg/L) is the concentration of dyes at reaction time, t (min).The photocatalytic activities of 2 has four Zn(II) centers, and both centers are in different coordination environments . These materials work well as functional photocatalysts for the degradation of antibiotics when exposed to visible light. At an apparent rate constant of 0.0320 min−1, the photocatalytic degradation efficiency of antibiotics over CPs was found to be over 82% in 45 min, which is similar to some of the best composite photocatalysts.In summary, we successfully synthesized three new CPs using a 3,5-di(1H-imidazol-1-yl)pyridine (dip) ligand and different carboxylic acids by using a hydrothermal technique. The results demonstrate that the use of different carboxylates shows that coordination ability, as well as metal centers with varying coordination environments, has a remarkable impact on the structures of the framework. Furthermore, the ligands’ various coordination modes may play an essential part in the development of the coordination framework. The structural studies demonstrate that