N′-[(1E)-1-(thiophen-2-yl)ethylidene]benzohydrazide (L) has been successfully designed and synthesized. The sensing studies of L demonstrated a ratio metric as well as turn-on-enhanced fluorescence and colorimetric response toward Fe3+ and Cu2+ ions, respectively and it was observed to be insensitive toward various metal ions. The Job plots revealed that the binding stoichiometry of L and metal ions is 2 : 1. In addition, density functional theory (DFT) results strongly suggested that L can be used as a powerful colorimetric sensor for the detection of Cu2+ ions. In vitro antimicrobial activities of L were evaluated by disk diffusion and results revealed good antibacterial activities against E. coli. Further, molecular docking was executed with DNA gyrase (PDB ID: 1KZN) of E. coli and the calculated interaction energy value was found to be −7.7 kcal mol−1. Finally, molecular docking, fluorescence, colorimetry and the HOMO–LUMO energy gap of the compound can provide new insights into developing drugs and detecting metals in biomolecules.Hydrazone–hydrazide-based linkers perform a crucial role in environmental as well as biological fields. Such linkers are employed to detect exact metal ions at a minute level; hence, numerous probes are available. Even though thiophene-based molecules have a unique position in the medicinal arena, only very few chemosensors are reported based on such a moiety. In this current work, a novel hydrazide–hydrazone-based fluorogenic molecule 5-bromo-2-hydroxy- N′-[(1E)-1-(thiophen-2-yl)ethylidene]benzohydrazide (L) has been designed and synthesized. The sensing studies of L showed a ratio metric & turn-on-enhanced fluorescence and colorimetric response toward Fe3+ and Cu2+ ions respectively.A novel molecule 5-bromo-2-hydroxy- PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>CH– functional group. Due to various medicinal properties such as antibacterial, antiviral, antifungal, anticancer and anti-inflammatory activities, such compounds have attracted substantial interest over many years in the design of novel chemical compounds.1–7 Moussa and co-workers reported a series of organic compounds containing the hydrazone moiety, which demonstrated high antioxidant activity against tyrosinase and cholinesterase.8 Rahim et al. designed and synthesized an aromatic hydrazide-based Schiff base targeting Alzheimer's disease that was shown to significantly inhibit acetylcholinesterase and butyrylcholinesterase compared to the standard physostigmine.9Hydrazide–hydrazones are a unique category of Schiff base compounds containing the –CO–NHN10 Fluorescent sensors have attracted extensive attention from researchers in the past few decades owing to their operational simplicity, high sensitivity, extreme selectivity, low instrumental cost, nondestructive sample analysis, etc.11 The signal of a fluorescent sensor is usually monitored as a change in its emission intensity, fluorescence lifetime, or a shift of emission wavelength to subtle environmental changes. On the other hand, the development of highly selective ratio metric and “turn-on” fluorescent probes have superior significance due to their capability of detecting metal ions even in living systems, which was found to display different emission behavior upon metal binding.12,13Moreover, chemosensors containing hydrazone–hydrazide moieties are considered potential candidates due to their high sensitivity and selectivity for detecting several cations and anions.14 It plays a ubiquitous role in many biochemical processes of plants and animals including oxygen transport, cellular metabolism, electron transfer and serves as an active site in myoglobin, hemoglobin, siderophores, and cytochromes.15 Especially, ferric ion (Fe3+) is responsible either for structural purposes or as part of a catalytic site in many proteins, lipids and enzymes.16 The deficiency of iron will lead to low oxygen delivery to cells, resulting in anemia, hemochromatosis, liver/kidney damage, diabetes, and cancer.17 Equally, excess iron in a living cell is considered a biohazard as it is prone to produce reactive oxygen species (ROS) through Fenton-reaction, which induces several serious diseases, such as Alzheimer's, Huntington's, and Parkinson's diseases.18 On the other hand, Cu2+ is the third most essential transition metal in the human body, which plays several important roles, for example, iron absorption, maintaining nerves, and blood vessels.19 Moreover, it acts as a catalytical co-factor in several metalloenzymes, for instance, superoxide dismutase, cytochrome c oxidase, and tyrosinase.20 However, the abnormal level of copper intake also causes destructive consequences to the body (WHO >2 ppm), such as irritation of the nose and throat, nausea, vomiting, and diarrhoea.21,22 On the contrary, copper deficiency in the human body leads to abnormal growth of bones. It turns toxic due to accumulation in cell membranes that lead to the disorder of cellular homeostasis, which causes oxidative stress accompanied by several neurodegenerative syndromes, ranging from Menkes, Wilson, hallucinations, depression, familial amyotrophic lateral sclerosis, schizophrenia, and Alzheimer's.23,24 Hence, the design and synthesis of new chromo-fluorogenic sensors for the detection of metals through simple synthetic mechanisms are of considerable importance for the environment and human health.Iron is one of the most abundant metals in the human body as well as in the Earth's crust.3+ and Cu2+ complexes. In addition to that, L was tested in vitro antimicrobial study against some Gram-negative, Gram-positive bacteria, and the fungus strain showed moderate inhibitory results. Finally, molecular docking methodology was used to study the molecular behavior of L with E. coli to identify their binding interactions.Therefore, the present study was designed to synthesize hydrazide–hydrazone-based compound L derived from the coupling of aromatic ketone and phenolic acids hydrazides containing bromo and hydroxyl groups. L possesses a tridentate –ONS– binding pocket . Density2.−1, using KBr disk), 1H, and 13C NMR spectra were recorded on a Bruker DPX-400 spectrophotometer using tetramethyl silane as an internal reference. NMR spectra were recorded on dimethyl sulfoxide (DMSO) solvent. Mass spectra were obtained from VG Micro mass 7070HS (EI) and HP1100MSD (LCMS) spectrometers. Steady-state fluorescence spectra were recorded on a HORIBA NanoLog spectrofluorometer. Time-correlated single photon counting (TCSPC) measurements using a HORIBA-Deltaflex were conducted for fluorescence lifetime measurements.All chemicals were purchased from Sigma-Aldrich and used as received without further purification. Infrared (IR) spectra were recorded on a Shimadzu (FTIR) Prestige-21 spectrophotometer (range: 4000–400 cm2.1.−1) , 3290s (–NH), 1637s (–CO), 1599s (–CN), 956s (–N–N). 1H NMR  , 11.25 , 8.03 , 7.65 , 7.59 , 7.57 , 7.13 , 7.00 , 2.37 . 13C NMR   in methanol (20 mL) was added to a solution of 2-acetyl thiophene  in methanol (10 mL) with continuous stirring. The resulting mixtures were stirred at 60 °C over a period of 6 hours . A yello) Fig. S1: ν 3477s) Fig. S2: δ 12.11) Fig. S3: δ 176.8+ Fig. S4: m/z 3372.2.In vitro antimicrobial action of compound L was assessed by agar disc diffusion technique.1 Mueller Hinton Agar (MHA) media was utilized as a control for bacteria analysis and Potato Dextrose Agar (PDA) media was utilized for fungal strain as already discussed and reported earlier.1 After preparation, the MHA and PDA media were nurtured for a day, and contaminations were tested. The test organism was inoculated by means of the sterile cotton bar on media after incubation. The sample discs were kept for 1 day for antibacterial assay at 37 °C and for 2 days at 26 °C for antifungal assay on pre-inoculated agar plates and aerobically incubated. DMSO was employed as a control. Each disc was introduced with 50 μL of the sample in DMSO having 300 μg of the synthesized compound, L. 50 μg per disc for the standard ceftriaxone and amphotericin B solutions in DMSO were loaded per disc for antibacterial and antifungal assays as a positive control, respectively. The diameter of the zone of inhibition (ZOI) in mm circling of the disc was assessed after the timely incubation for antibacterial and antifungal assay. In this research, two Gram-positive Staphylococcus aureus (cars-2) and Bacillus megaterium (BTCC-18), two Gram-negative Escherichia coli (carsgn-2) and Salmonella typhi (JCM-1652) bacteria, as well as two fungal strains Trichoderma harzianum (carsm-2) and Aspergillus niger (carsm-3) were utilized.2.3.E. coli (PDB ID: 1KZN) was obtained in pdb format from the online protein data bank (PDB) database. The structure was verified, and energy minimization was performed with the Swiss-Pdb Viewer software packages (version 4.1.0),25 since the crystal structure contains a variety of issues related to improper bond order, side chains geometry, and missing hydrogen atoms. Prior to docking, all the heteroatoms and water molecules were removed from the crystal structure using PyMol (version 1.3) software packages.26 Both the structures of the proteins and ligands were saved in .pdbqt format by PyRx 0.8 for docking analysis.27 The grid box that encloses amino acids domain involved in the binding active sites, had the dimension of 25 × 25 × 25 Å3 (x × y × z) and center of 19.53 × 19.16 × 43.28 Å3 (x × y × z). The docked conformation of the respective protein conformer with the lowest binding free energy was analyzed using PyMOL Molecular Graphics System (version 1.7.4) and Accelrys Discovery Studio 4.1.28The 3D crystal structure of 3.3.1.3+, Mn2+, Pb2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Na+, K+, Mg2+, Ca2+, and Al3+ was achieved in DMSO as demonstrated in 2+ enhanced significantly produced an intensive color change, while the rest of the metal ion solutions showed a very mild alteration or almost no change in color. Hence, the combination of the ligand with copper ions was further investigated by spectroscopic and theoretical means. The UV-vis absorption spectra of the ligand in DMSO showed an absorbance maximum at 330 nm due to a symmetry-allowed π → π* transition rather than a symmetry-forbidden n → π* transition. In addition, two isosbestic points were observed at 290 and 346 nm in the colorimetric titration of the ligand with Cu2+ as shown in 2+ was obtained using the calibration curve of absorbance versus concentration, which was found to be as low as 2.35 μM.The recognition profiles of L (10 μM) toward different metal cations (1 eq.) were also investigated by absorption spectroscopy. The selectivity of L towards various cations Cr3.2.3+. As evident from 3+, a new significant broad fluorescence emission at about 550 nm was clearly observed, whereas Cr3+, Mn2+, Pb2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Na+, K+, Mg2+, Ca2+, Al3+ and Bi2+ showed no emission band at this wavelength. These results indicated that L could function as a fluorescent “turn-on” type sensor for Fe3+ ions. It can be assumed that the emergence of the new fluorescence emission might demonstrate the coordination process of Fe3+ with L, which is also supported by the generation of a new absorption band at 355 nm in the UV-vis spectrum of L + Fe3+ when compared with that of L (λem − λabs/ex = 195 nm) of L + Fe3+ enables the clear separation of the excitation and emission bands, which is highly desirable for the fluorescent detection of metals. For better investigation, we have carried out the fluorescence titration of L with the subsequent addition of Fe3+ and found that the emission intensity at 550 nm gradually increased. The ratio metric and fluorescence enhancement of L witnessed the visual detection of Fe3+ ions when the samples were exposed to a UV lamp. The quantum yields of L increased from 0.0019 to 0.052 upon binding of Fe3+. Accordingly, L could be utilized as an excellent turn-on fluorescence chemoreceptor for Fe3+ ions  disappeared with the addition of metals  of the receptor L, that inhibits the non-radiative transitions such as –CN isomerization. The –CN isomerization inhibition process induced by the Fe3+ binding warrants the chelation-induced enhanced fluorescence (CHEF) effect.33 Thus, the fluorescence enhancement of L upon binding to Fe3+ might be due to CHEF effect.3.4.2+ ion, it was employed to detect concentration of Cu2+ in tap water samples. In order to verify the accuracy of the established procedure, recovery experiments were carried out by spiking the tap water samples with different concentrations of Cu2+ and L in DMSO (2 mL) before any pretreatment.34 The change in absorbance of L with the above water samples was tested by adding 20, 40, and 80 μM of Cu2+ individually. Each sample was analyzed three times in H2O/DMSO . The results are tabulated in To test the reliability of L in a real sample with Cu3.5.In vitro sensitivities of two Gram-positive and two Gram-negative bacteria including two fungal strains against the synthesized compound were assessed by agar disc diffusion technique.1,35 The formation of the diameter of ZOIs in mm by the synthesized analogue is shown in E. coli bacteria compared to other bacterial and fungal strains. The outcomes represented in E. coli, with a mean ZOI of 12 mm diameter at 300 μg mL−1. While, L had a low mean ZOI for T. harziana, and A. niger, suggesting that had a low antifungal activity effect.3.6.36,37Molecular docking, a subfield of computational chemistry and bioscience, is a powerful tool to investigate and provide several quick insights into ligand–receptor interactions in order to facilitate the design of potential drugs.E. coli was performed. Thus, to get the binding conformations of these compounds, L was docked in the active site of E. coli DNA Gyrase (PDB ID: 1KZN) using the PyRx 0.8. The most stable anchoring conformations of these compounds along with interacting residues are shown in 29To investigate and compare the antimicrobial activity of the synthesized compound with experimental data, a docking analysis of L against 1KZN were −7.7 kcal mol−1, whereas the binding energy for Ciprofloxacin was reported −9.2 kcal mol−1.38 In L-1KZN, L formed two conventional hydrogen bonds (2.63 and 3.00 Å) of O–H–O–C with active site residues GLU50 and THR165. Pi-anion, pi-sigma and pi-alkyl bonds were also noticed with GLU50 (4.23 Å), VAL43 (4.62 Å), ALA47 (5.43 Å), VAL71 (5.47 Å), ILE78 (5.07 Å), VAL167 (5.06 Å), PRO79 (4.82 Å), and THR165 (3.75 Å) respectively. It was also noted that an electrostatic bond was found between N and residue ASP73 as shown in 1KZN and showed strong interactions with GLU50, THR165, and ASP73 of DNA gyrase enzyme (PDB ID: 1KZN) as shown in 20,39 Thus, computational results are in good agreement with in vitro experimental data /LANL2DZ for C, H, N, S, O, and metal ions, where for the complexes multiplicity were 2.3+ and Cu2+.40–43 The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of L were located within mostly on the whole π-moiety of thiophene as well as the –CO–NHNCH– moiety with a HOMO–LUMO gap of 2.507 eV, which indicated the transition of π to π* of the thiophene unit. As evident from UV-visible and fluorescence titration spectra, the stoichiometry of L with Fe3+ and Cu2+ were confirmed strongly as 2 : 1 and hence, the optimization of the metal complex was carried out with 2 : 1 coordinated complexes. In the L-Fe3+ complex, the HOMO is situated completely on the whole ligand L, whereas LUMO is concentrated over Fe3+ ions with a band gap of 1.095 eV . That's why L can act as an excellent chemosensor for the detection of Cu2+ in the environment. Also, L may be applied for the detection of biologically important metal ion Fe3+ in the bioimaging of live cells. Thus, due to the wide application of such hydrazide–hydrazone compounds and their simple, low-cost synthetic procedure, such compounds can be a target for drug design.In summary, a novel hydrazide–hydrazone-based chemosensor has been synthesized and characterized successfully by FT-IR, Conceptualization, M. A. M. P. and S. A. R.; methodology, M. A. M. P., and S.A. R.; software, S. A. R. and M. A. M. P.; validation, A. K. M. R. U., M. A. M. P. and J. U.; formal analysis, M. A. M. P., S. A. R. and W. E. G.; investigation, S. A. R., W. E. G., A. A. and M. A. H.; resources, M. A. M. P., M. A. H., M. K. and J. U.; data curation, M. A. M. P., W. E. G., A. A. and S. A. R.; writing – original draft preparation, S. A. R., M. A. M. P. and A. K. M. R. U.; writing – review and editing, all authors; visualization, M. A. M. P., M. K. and J. U.; supervision, M. A. M. P. and J. U. All authors have read and agreed to the published version of the manuscript.The authors declare no competing financial interest.RA-013-D3RA04364A-s001


Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species’ eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These “unlighted” species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, The online version contains supplementary material available at 10.1007/s10886-023-01417-2. Summer firefly flashes are a nostalgic reminder of nature’s wonder. The bioluminescent signals of these charismatic beetles (family: Lampyridae), emitted from dusk into the night, are renowned for their function in mediating the finding, identifying, and choosing of mates Lloyd . HoweverAside from the lack of light organs, evidence for diurnally active species’ use of volatile pheromones comes from (i) behavioral assays where males were attracted to a petri dish containing a female, sometimes even after the female had been removed Lloyd , (ii) coPhotinus corruscus  or a hexane rinse, as exo,exo-2,3-camphanediol. However, the synthetic racemate of this compound did not elicit attraction or mating behavior in laboratory behavioral assays. Additionally, these previous studies focused on close-range, contact interactions, such as those mediated by CHCs, which are relatively nonvolatile, making them unlikely candidates for long-range attractant pheromones  and single sensillum recordings (SSR), we located an odorant receptor neuron (ORN) in males that responds specifically to this compound. To our knowledge, this is the first sex attractant pheromone identified from any firefly species.Here, using coupled gas chromatography - electroantennographic detection (GC-EAD) and coupled gas chromatography-mass spectrometry (GC-MS), we identified a female-produced compound from headspace volatiles of female P. corruscus were collected during March and April 2021 on the sides of large-diameter trees in Lewisburg, PA . At this point in the season, they were likely emerging from winter diapause  sealed with a reusable foam plug . Because winter fireflies are one of the few firefly species that are suspected to feed as adults, potentially consuming nectar or sap .To capture firefly-emitted compounds, collections of headspace volatiles were conducted using wide-mouth 250 mL Teflon jars , with the screw cap lids fitted with Swagelok bulkhead unions  to connect inlet and outlet tubes. Air, purified by passage through granulated activated charcoal , was pulled through the system by vacuum at 250 mL/min. Volatiles were adsorbed onto ~50 mg of thermally desorbed activated charcoal  held between glass wool plugs in a short piece of glass tubing. Aerations were conducted in a controlled environment insectary room under ReptiSun 100 UVB lights  at 24 ± 1 °C with a 16:8 h L:D cycle to simulate summer daytime conditions. Volatiles were eluted from the charcoal with 500 μL of dichloromethane  and 5 of males  and column .To identify firefly-emitted volatile compounds, the aeration extracts were initially analyzed by GC-MS in splitless mode with an Agilent 7820A GC interfaced to an Agilent 5977E mass selective detector . A DB-5MS column was used , with a temperature program of 40 °C for 1 min, then ramping at 10 °C /min to 280 °C, followed by a hold for 10 min. The injector and transfer line temperatures were 250 °C and 280 °C, respectively. Mass spectra were collected in electron ionization mode (EI) at 70 eV, with a scan range of P. corruscus antennal preparation.Subsequently, aeration extracts and synthetic standards were analyzed by GC-EAD with an HP 5890 Series II GC equipped with a DB-5MS column . The injector and detector temperatures were 250 °C and 280 °C, respectively, with a column head pressure of 200 kPa. The temperature program was 50 °C for 1 min, then ramped at 20 °C/min to 280 °C, then held for 10 min. The column effluent was split to the flame ionization detector  and a heated transfer line (260 °C) with a glass X-cross, with additional helium being added at the fourth arm at 3 ml/min as makeup gas in order to maintain the flow rate to both the FID and EAD. Effluent passing through the transfer line was directed into a humidified airstream at 650 ml/min in a glass tube (15 mm ID), which then passed over the P. corruscus were prepared by removing an entire antenna with a razor blade followed by removing a portion of the distal tip. The antenna was then placed between two glass capillary electrodes filled with saline solution . Electrical connection was made with a 0.2 mm diameter gold wire in each capillary, attached to a custom-built amplifier. Signals from the FID and the amplifier were recorded in tandem using Peak-Simple software . All GC-MS and GC-EAD files derived from sample aerations are available on Figshare (10.6084/m9.figshare.21648497).Antennae of 2Cl2 as possible. The concentrated sample in ~0.1 mL pentane was then loaded onto a column of silica gel  in a disposable pipette, pre-wetted with pentane. The column was eluted sequentially with 2 × 0.5 mL pentane, 2 × 0.5 mL 25% ether in pentane, and 2 × 1 mL ether, collecting each as a separate fraction. The compound of interest with an apparent molecular ion at m/z 168 started eluting in the 2nd 25% ether fraction (Frac. 4), with the bulk eluting in the first 100% ether fraction.To isolate the compound of interest identified by GC-EAD, five aeration extracts from females in dichloromethane (~2.5 mL) were first combined and concentrated to ~0.1 mL under a gentle stream of nitrogen, and 0.5 mL pentane was added. The sample was blown down again, and the procedure was repeated to remove as much of the CH2 for 1 h to reduce any carbon-carbon multiple bonds. The mixture was then filtered through a small pad of celite, rinsing with ether, concentrated to ~100 μL, and an aliquot was analyzed by GC-MS. Approximately half of the remaining H2-reduced sample was diluted with 0.5 mL ether, and ~ 2 mg LiAlH4 was added to reduce any carbonyls. The mixture was stirred for 1 h at room temp, then quenched by careful addition of 0.1 mL aqueous 1 M HCl, followed by 0.5 mL saturated brine. The mixture was vortexed, and the ether layer was removed and dried over anhydrous Na2SO4, and an aliquot was analyzed by GC-MS. To test the effects of oxidation, a second aliquot of fraction 4 (eluted with 25% ether in pentane) was concentrated to ~10 μL, then diluted with 0.1 mL CH2Cl2, ~2 mg finely powdered pyridinium dichromate was added, and the mixture was stirred for 1 h at room temp. The mixture was then diluted with 0.5 mL ether, and filtered through a plug of celite. An aliquot was analyzed by GC-MS.To aid in the identification of the compound, a series of micro-derivatization experiments were conducted. An aliquot of the 100% ether fraction was diluted 1:1 with pentane, ~ 1 mg of 10% Pd on carbon was added, and the mixture was stirred under H2SO4 and concentrated by rotary evaporation under partial vacuum. Flash and vacuum flash chromatography were carried out on silica gel . TLC analyses were conducted on aluminum-backed sheets of analytical silica gel 60 F254 , and compounds were visualized by spraying with 10% phosphomolybdic acid in ethanol and heating. Yields are reported as isolated yields of chromatographically pure products unless otherwise noted. Mass spectra were obtained with an HP 6890 GC (Agilent Technologies) equipped with a DB-17 column , coupled to an HP 5973 mass selective detector in EI mode (70 eV) with helium carrier gas. Purity was assessed by gas chromatography with an HP 5890 GC equipped with a DB-5 column (30 m × 0.25 mm × 0.25 μm film), unless otherwise noted. Isomeric and enantiomeric purity were assessed by gas chromatography with an HP 5890 GC equipped with a chiral stationary phase β-cyclodextrin column . Nuclear magnetic resonance (NMR) spectra were recorded as CDCl3 solutions on either a Bruker Avance 500 or Bruker NEO 400 spectrometer. Chemical shifts are reported in ppm relative to CDCl3 .All solvents were Optima grade (Fisher Scientific) unless otherwise noted. Anhydrous diethyl ether stabilized with butylated hydroxytoluene was purchased from Fisher Scientific. Solutions of crude reaction products were dried over anhydrous Na4  was added to a solution of (S)-(−)-camphorquinone  in 2.5 mL dry ether. The mixture was stirred 1 h at room temp, then quenched with 1 mL of 1 M HCl. The mixture was then diluted with 10 mL brine and extracted with ether. The ether solution was dried and concentrated, and the residue was fractionated by flash chromatography on silica gel, eluting with 25% EtOAc in hexane, achieving a partial separation of the isomers. The fractions were checked by GC on a DB-5 column, matching the retention time of one hydroxycamphor isomer with that of the insect-produced compound. An aliquot was then reanalyzed by GC-MS on a DB-17 column, matching both the retention time and the mass spectrum of one of the isomers with those of the insect-produced compound.LiAlH1H-NMR spectral data -camphorquinone , diethyl ether (0.5 mL), and methanol (0.5 mL). The flask was cooled to 0 °C followed by the addition of NaBH4  in one portion. The reaction was stirred at 0 °C for 30 min, then quenched with water (167 μL) and diluted with brine (300 μL). The mixture was extracted with diethyl ether (3 × 1 mL), and the organic extracts were combined, washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure to yield a crude white solid composed of a 65:35 mixture of isomers. This crude material was purified by vacuum flash column chromatography (20% EtOAc/Hex) to yield 59 mg (73%) of a 65:35 mixture of isomers (1S)-exo-2 and (1S)-exo-3. (1S)-exo-2: 1H NMR : δ 3.73 , 3.23 , 2.07 , 1.97 , 1.63 , 1.47–1.30 , 0.96 , 0.92 , 0.90 . 13C NMR  δ 220.52, 77.26, 57.02, 49.22, 46.72, 28.52, 25.11, 20.95, 20.00, 8.96. (1S)-exo-3: 1H NMR : δ 3.53 , 3.23 , 2.14 , 1.89 , 1.82 , 1.47–1.30 , 1.01 , 1.00 , 0.90 . 13C NMR  δ 219.27, 79.35, 58.58, 49.15, 46.51, 33.77, 21.12, 20.28, 18.78, 10.25. (1S)-exo-2 and (1S)-exo-3: EI-GC/MS m/z (%): 168 , 153 (6), 140 (9), 125 (41), 107 (11), 100 (13), 84 (92), 83 (100), 71 (22), 70 (33), 69 (42), 57 (20), 55 (53), 43 (22), 41 (46).A dry flask flushed with Ar was charged with -camphorquinone , and water (1.8 mL) and heated to 100 °C. The heterogeneous yellow mixture was briefly stirred, then zinc dust  was added in portions over 1 min. The reaction was stirred for 20 min at 115 °C, becoming colorless. The reaction was allowed to cool, then diluted with water (2 mL) and extracted with diethyl ether (2.5 mL). The aqueous layer was extracted with additional diethyl ether (2.5 mL), and the combined organic layer was sequentially washed with sat. aq. NaHCO3 (1 mL) and brine (1 mL), dried, concentrated, and the crude material was purified by vacuum flash chromatography (20% EtOAc/Hex) to yield 81 mg (80%) of a 56:44 mixture of the two endo isomers (1S)-endo-2 and (1S)-endo-3. (1S)-endo-2: 1H NMR : δ 4.20 , 3.35 , 2.26 , 1.93 , 1.70 , 1.67 , 1.38 , 0.98 , 0.90 , 0.85 . (1S)-endo-3: 1H NMR : δ 3.84 , 3.35 , 2.24 , 1.96 , 1.38 , 1.03 , 0.95 , 0.90 . (1S)-endo-2 and (1S)-endo-3: 13C NMR  δ 220.95, 219.53, 78.73, 74.40, 59.40, 58.41, 50.22, 48.58, 43.08, 43.01, 32.48, 24.99, 24.62, 19.94, 19.32, 18.76, 17.84, 12.91, 9.26. EI-GC/MS m/z (%): 168 , 153 (14), 139 (11), 135 (7), 125 (32), 109 (14), 107 (11), 95 (29), 84 (75), 83 (89), 71 (63), 70 (100), 69 (52), 55 (50), 44 (40), 43 (67).A flask was charged with acetic acid , (1S)-camphorquinone  and dry THF (200 mL), then cooled to −78 °C. L-Selectride  was added over 1 h, and the mixture was stirred an additional 5 min. The reaction was quenched by addition of 3 M HCl in methanol (16 mL) over 10 min at −78 °C, then allowed to warm to room temperature. The resulting mixture was diluted with brine (500 mL) and the products were extracted with dichloromethane (4 × 200 mL). The combined organic layers were dried, concentrated, and purified by vacuum flash chromatography (5% - 25% EtOAc/Hexanes). The fractions rich in the desired product were concentrated and subjected to flash column chromatography (20% EtOAc/Hexanes) to partially separate the isomers of hydroxycamphor. The resulting material was sublimed with a Kugelrohr distillation apparatus , yielding 397 mg (7%) of (1S)-exo-2, 92% isomerically pure (NMR). Impure fractions were consolidated and purified again by flash chromatography to yield an additional 298 mg of (1S)-exo-2, ~95% isomerically pure. 1H NMR  δ 3.72 , 3.28 , 2.06 , 2.02–1.91 , 1.62 , 1.46–1.29 , 0.96 , 0.91 , 0.89 . 13C NMR  δ 220.51, 77.26, 57.01, 49.22, 46.71, 28.51, 25.10, 20.94, 19.98, 8.95. EI-GC/MS m/z (%): 168 , 153 (2), 140 (9), 125 (42), 107 (10), 100 (14), 84 (92), 83 (100), 69 (38), 57 (19), 55 (51), 43 (14), 41 (37).The following procedure was adapted from Kouklovsky et al. . A dry fendo-hydroxycamphor endo-2. However, in our hands, and based on other published reports - and (1S)-camphor (0.33 mmol) was used in the Davis oxidation with the intention of producing a differentiable mixture of endo-hydroxycamphor enantiomers. After discovering that the insect-produced compound and the minor enantiomer from the Davis oxidation product matched, a larger scale reaction from (1S)-camphor (>98%) was run as follows. A dry flask flushed with Ar was charged with THF (40 mL) and potassium hexamethyldisilazide , then cooled to −78 °C. (1S)-Camphor  was added in portions over 1 min and the mixture was stirred 30 min at −78 °C. A solution of the Davis reagent, 2-(phenylsulfonyl)-3-phenyloxazaridine  was added dropwise over 20 min. The reaction was stirred at −78 °C for 55 min. Upon completion, the reaction was quenched with sat. aq. NH4Cl (10 mL) at −78 °C and allowed to warm to room temperature. Brine (30 mL) and diethyl ether (30 mL) were added, and the layers were separated. The aqueous layer was reextracted with diethyl ether (30 mL), then the combined organic layers were washed with brine (20 mL), dried, and concentrated. The resulting cloudy yellow oil was dissolved in 4:1 dichloromethane:hexanes and partially purified by vacuum flash column chromatography (20% EtOAc/hexanes). The fractions enriched in the desired product were concentrated and repurified by flash column chromatography (25% EtOAc/hexanes). The product was then purified further by Kugelrohr distillation  to yield 182 mg of product (49%), containing ~85% of the desired insect-produced exo-hydroxycamphor isomer (1S)-exo-2 and 15% of the endo-hydroxycamphor isomer (1S)-endo-2. EI-GC/MS m/z (%): 169 (4), 168 , 153 (2), 140 (9), 125 (43), 107 (11), 100 (15), 84 (93), 83 (100), 69 (38), 57 (18), 55 (50), 43 (14), 41 (35).In a preliminary synthesis, a 2:1 mixture of  with 100 μL of a 10 mg/mL solution of 92% pure synthetic hydroxycamphor ((1S)-exo-3-hydroxycamphor) in pentane (1 mg pheromone). Controls consisted of septa dosed with 100 μL pentane. Control and experimental lures were held in separate glass bottles at −20 °C until field deployment. Lures were deployed inside a wire mesh tea strainer suspended from a paper-clip hook in the middle of the cake round. A coat of TangleTrap  was applied to the remaining cake round surface (front side only) to provide a sticky surface in which attracted males, landing nearby and scrambling toward the pheromone source, would adhere. To avoid potential bias in TangleTrap application based on trap treatment status, the sticky coating was applied prior to trap assignment as either a control (solvent only) or experimental (solvent + pheromone) trap. Treatment  of the first trap in the transect was determined using a random number generator , and the status alternated along the transect. After 24 h, the individuals stuck in each trap were removed, preserved in 95% EtOH, and identified to sex by examination of external morphology under a dissecting microscope  and Middlebury College in Middlebury, VT . At each site, 10 sticky traps were deployed every 20 m in a single transect, with the sticky surface south-facing. Traps consisted of 10-in. cardboard cake rounds covered with transparent packing-tape to repel moisture and secured to the top of a 4-ft fence post , mounting , or copulating (attempted aedeagus insertion), as well as total time in contact , was recorded for both the control and experimental septa.Two-choice assays were conducted between control (solvent only) and experimental (solvent + pheromone) septa in laboratory assays with male https://selower.github.io/Winter_firefly_pheromone_project/).For field bioassays, because catch data were not normally distributed, the attractive effect of the pheromone relative to solvent controls was assessed using a one-sided Mann Whitney Wilcoxon test in R . Coated antennae were then imaged using a Vega 3 LMU Scanning Electron Microscope  with an accelerating voltage of 5 mV and a beam intensity of 8.From P. corruscus were collected in pheromone-baited traps in Middlebury, VT in May 2022. Collected fireflies were housed under ambient light conditions in plastic vials with a moistened Kimwipe 1–4 d before being prepared for electrophysiology. Fireflies were immobilized on a microscope slide with the ventral side up using double-sided tape. Additional strips of double-sided tape were used to immobilize the base and distal ends of the antennae, leaving both the head and middle segments accessible to electrodes. The preparation was mounted under a BX51WI microscope  and kept under a continuous 20 mL/s flow of humidified air using a CS-55 stimulus controller . Sharp glass electrodes filled with sensillum lymph Ringers solution .To test the detection limits of pheromone-sensitive sensilla, different doses of pheromone were delivered to the sensillum using cartridges made from glass Pasteur pipettes and 1 mL pipette tips. Pheromone solutions in pentane (pheromone doses of 0.1–100 μg) or pentane control were applied to the inside of a Pasteur pipette, which was left open for 5 min to allow for pentane evaporation before being sealed with a 1 mL pipette tip and parafilm squares at either end. Pheromone cartridges were inserted into the continuous stream of humidified air and 16 mL/s charcoal-filtered air switched from the blank cartridge to the pheromone cartridge for 1 s using the CS-55 stimulus controller. Each recording consisted of multiple 10 s traces . Neuronal responses to pheromone stimulation were collected and analyzed manually using Axoscope 10 software (Molecular Devices). The baseline firing rate was determined for each neuron during the 1 s before each stimulus, and the stimulus firing rate was counted in a 500 ms window between 150 and 650 ms of the stimulus delivery to account for the delay in the airflow delivery system. The change in firing frequency (∆ spikes/s) was calculated by subtracting the baseline firing frequency from that of the stimulus. Data from 8 basiconic sensilla from 5 P. corruscus fireflies collected from February through June of 2021 and 2022 in Union and Montour Counties, Pennsylvania  at m/z 168  the unknown was unchanged by catalytic hydrogenation, indicating the absence of carbon-carbon pi bonds; (ii) the unknown seemed to be reduced with LiAlH4, such that the unknown peak disappeared from the GC trace, but the product(s) were not found; and (iii) when an aliquot of the insect extract was oxidized with pyridinium dichromate  by a match with the NIST mass spectral database, increased in size. The identification was confirmed by matching the retention time and mass spectrum with those of an authentic standard of camphorquinone 1. The facts that the unknown had a molecular weight that was two mass units higher than that of camphorquinone 1, and that PDC oxidation converted the unknown to camphorquinone 1, indicated that the unknown had to be a hydroxycamphor isomer -camphorquinone 1 with LiAlH4 yielded a mixture of 4 isomers -camphorquinone. The remaining uncertainties were the relative positions of the ketone and hydroxyl groups, and whether the hydroxyl group was on the same face as the geminal dimethyl bridge (exo), or the opposite face (endo), i.e., the four possible isomers shown in Fig. In a “quick and dirty” proof of the unknown’s structure, partial reduction of  and NMR of the standards  were matched with the results from analyses of the same samples on a chiral stationary phase Cyclodex B column . Having unambiguously identified each of the synthetic standards, the insect-produced compound was then analyzed under the same conditions, to match it with one of the isomers.exo-hydroxycamphor isomers, by reduction of (1S)-camphorquinone ((1S)-1) with NaBH4 -exo-2 and (1S)-exo-3. This composition is in accord with the literature -endo-2, a 2:1 mixture of (R)- and (1S)-camphor 4 was oxidized with the Davis reagent (2-(phenylsulfonyl)-3-phenyloxaziridine -exo-2 (NMR assignment and reaction outcome supported by Piątek and Chapuis (R)-endo-isomer reported by Davis et al. (S)-exo-2 from (1S)-camphor 4 co-eluted with the insect produced compound on the chiral Cyclodex B GC column. Taken together, this indicated that the insect-produced compound was likely to be (1S)-exo-2.In pursuit of the ine Fig. . Two key Chapuis  and Neis Chapuis ) rather s et al. , and (2)endo-hydroxycamphor candidates remaining to be synthesized, a Zn dust/AcOH reduction of (1S)-camphorquinone was performed -endo-3. Even though the identification of each regioisomer on the chiral GC chromatogram could have been difficult because the product mixture is composed of ~equal proportions, the two isomers coincidentally co-eluted and eluted at a different time relative to the insect-produced compound, confirming that neither endo isomer could be the insect-produced compound. This allowed us to unambiguously identify the unknown as (1S)-exo-2, formally, (1S)-exo-3-hydroxycamphor -3-hydroxy-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one).With the two med Fig. c. This sS)-exo-2 was carried out by reduction of (1S)-camphorquinone 1 with L-Selectride. Unlike syntheses reported in the literature that claimed a completely stereoselective transformation -exo-2 and (1S)-endo-2, 45% yield, inclusive) was obtained after chromatography. Although literature reports claimed that the isomers are inseparable by column chromatography -exo-2 in up to 95% isomeric purity (across multiple experiments), with the remainder being (1S)-endo-2. Finally, the crystalline material was sublimed in a Kugelrohr distillation apparatus, at no detriment to isomeric purity.A larger scale, stereoselective synthesis of compound . The longer multiporous basiconic sensillum -exo-3-hydroxycamphor) is a sex attractant pheromone of P. corruscus. In a previous attempt to identify the sex pheromone of P. corruscus, Ming and Lewis . Future studies of the range and importance of the various signals associated with mating will elucidate their roles in this species. m Lloyd . Our perPhotinus corruscus is hypothesized to be a species complex rather than a single species ESM 2(XLSX 18.0 kb)


Seeds were primed for 12 h at 25 ± 2°C, by soaking in an aerating solution [urea (20 g L−1) + FeSO4.7H2O (50 ppm) + ZnSO4.7H2O (50 ppm) + silicon (20 mg L−1)]. Seed weight-to-solution volume ratio was 1:5 (kg L−1). A pot experiment was also conducted to examine the effect of priming on root growth. Overall, combined seed priming and foliar application induced a positive impact on physiological traits and attributes. Maximum chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid concentrations , membrane stability index (77.31%), superoxide dismutase and peroxidase activity (0.174 and 0.375 Unit mg−1 protein), 1,000-grain weight (35.30 g), biological yield, grain yield , and minimum malondialdehyde concentration (3.91 µg g−1 FW) were observed in seed priming combination with ZnSO4 foliar application. The highest glycine betaine concentration (6.90 mg g−1 DW) and proline (972.8 µg g−1 FW) were recorded with the co-application of seed priming and foliar urea spraying. Foliar application of ZnSO4, FeSO4, and urea drastically enhanced grain Zn (29.17%), Fe (19.51%), and protein content (increased from 11.14% in control to 12.46% in urea foliar application), respectively. Compared to control, seed priming increased root length, root volume, and dry mass root by 8.95%, 4.31%, and 9.64%, respectively. It is concluded that adequate Zn, Fe, silicon, and N supply through seed priming and foliar applications of these compounds at the terminal stage of rainfed wheat alleviates drought stress and improves GY and biofortification.Seed priming and foliar application are two crop management practices that can increase grain yield and quality. The research aimed to assess the influence of seed priming and foliar application on rainfed wheat. Two field experiments with two seed priming rates (control and priming) and five foliar applications [control, urea (4%), silicon (4 mM), FeSO BecauseSeveral studies have examined wheat priming and foliar applications of nutrients . Neverth22.12.1.1The field experiment was conducted at the University of Kurdistan’s research farm, which is situated on the Dehgolan plain  during the wheat-growing seasons of 2021–2023. 2.1.24.7H2O (0.6%), and ZnSO4.7H2O (0.4%)]. Each plot was 2.9×8 m2, and the distance between plots and blocks was 1 m and 2 m, respectively.In the two growing seasons, this study was conducted in factorial with four replications utilizing a randomized complete block design (RCBD) to assess the effect of seed priming and foliar application on rainfed wheat (Cv. Baran). The experimental factors included two priming levels (control and priming with) and five foliar applications . The ratio of seed weight to solution volume was 1:5 (kg L−1). During the soak, an aquarium pump supplied aeration. To ensure clump-free sowing of the seeds, the seeds were dried in the shade after being removed from the solution. Two foliar applications were performed, first at the anthesis/Z61  after separating flag leaves from plots to study this parameter.2.1.4.1A method based on 2.1.4.2Based on 2.1.4.3The GB was extracted from dry flag leaves using hot distilled water (70°C). Dry flag leaves were soaked in distilled water for 48 h and then shaken vigorously to measure GB. 2N HCl and potassium tri-iodide solution were added to the extract (0.25 ml). A 90-min ice bath was used to cool the contents after shaking. Following this, 20 mL of 1,2-dichloromethane (cooled at −10°C) and 2 mL of ice-cold distilled water were added. There were two layers formed from the mixture; 365-nm optical density measurements were performed after discarding the upper aqueous layer .2.1.4.4g for 5 min at 4°C.The 2.1.4.5Superoxide dismutase activity was determined based on 2.1.4.6−1 cm−1).Peroxidase activity was determined based on 2.1.4.7To determine the MSI, leaf discs were cut with scissors and inserted into glass vials. The samples were washed twice with double-distilled water for 5 min each. Following draining the water from each vial, 10 mL of double-distilled water was added. The vials were then shaken . The electrical conductivity was measured using a conductivity electrode. The conductivity was measured after 60 min of hot water bathing all vials. According to 2.1.4.82 of each plot in the center and expressing it at 12% moisture content. Divide GY by biological yield (BY) at maturity to calculate the harvest index (HI). We also measured the kernels per spike at maturity and the 1,000-grain weight.Grain yield at maturity was determined by cutting 3 m2.1.4.93 solution to determine Fe and Zn content. Atomic absorption spectrometry was used to detect Fe and Zn in grains  HNOn grains .2.1.4.10To measure grain protein content, the N concentration was initially measured with the Kjeldahl method. By multiplying the grain N content by the N-to-protein conversion factor (5.7), the grain protein content was calculated .2.1.5p ≤ 0.05  + FeSO4.7H2O (50 ppm) + ZnSO4.7H2O (50 ppm) + silicon (20 mg L−1)]. As described in section 2.1.2, the priming of seeds was done the same way in the field experiment. First, plastic pots (12 in diameter and 100 cm in height) were filled with 15 kg of soil . The pot experiment was conducted with soil obtained from the field.  of soil Table 1.2.2.22 mesh). As the sieve was suspended over a water bath, it was repeatedly shaken until soil was removed from the roots. The remaining soil materials were manually removed from the sieve. The measured root length, root volume, and root DM were then averaged. Root length was calculated using a ruler from the base to the tip, and total measurements were taken. Root masses were placed in a water-filled measuring cylinder to determine root volume. Water level increase was measured as cm3 pot−1. For each pot, roots were oven dried for 48 h at 72°C, and root DM was calculated as g pot-1.At maturity, plant dry mass (DM) and grain weight were measured in grams for each pot in this experiment. The pots were removed from the ground after harvesting the aboveground part at maturity in order to measure root length, root volume, and root dry weight. For 12 h, the pots were placed in water-filled barrels. Roots were stirred and poured into a sieve  was used to analyze the data using a completely randomized design (ANOVA). To compare the means, Duncan’s multiple range test was used at a p ≤ 0.05 .33.1A combined ANOVA for traits revealed that there was a significant year, seed priming, and foliar application effect for almost all traits Table 3.3.24 > silicon > FeSO4 > urea > control foliar application. Foliar application of ZnSO4, silicon, FeSO4, and urea at seed-primed treatment increased total chlorophyll content by 26.97%, 21.25%, 12.07%, and 7.99%, respectively, compared to the control. Furthermore, silicon, ZnSO4, FeSO4, and urea applied as foliar treatments at non-primed seed treatment increased Chl T by 17.48%, 16.83%, 12.08%, and 8.23% in comparison with the control (−1 FW) was recorded with seed priming and ZnSO4 foliar application, which was significantly higher compared to other treatments  at foliar application of urea, ZnSO4, silicon, and FeSO4 and no foliar application (Proline concentration was 6.65% higher in the first growing season than in the second growing season Table 3.3.4−1 DW) than in the 2022–2023 growing season (5.86 mg g−1 DW). The highest GB concentration (6.71 mg g−1 DW) was observed at the co-application of seed priming and foliar spraying of urea. Foliar application of urea, ZnSO4, silicon, and FeSO4 compared to control at seed priming treatment elevated GB concentrations by 19.22%, 15.86%, 14.31%, and 10.06%, respectively (A higher concentration of GB was observed in the 2021–2022 growing season (6.08 mg g3.54 and silicon resulted in a greater reduction in MDA concentrations than other foliar applications. The highest MDA concentration (6.19 µg g−1 FW) was detected in the control foliar application and non-primed seed. Foliar application of ZnSO4, silicon, FeSO4, and urea compared to control at seed priming treatment reduced MDA concentrations by 30.61%, 28.80%, 17.40%, and 11.17%, respectively  was observed in seed priming in combination with ZnSO4 foliar application. This was statistically similar to the foliar application of silicon (0.367 Unit mg−1 protein) at seed priming treatment. In seed priming treatment, foliar application of ZnSO4, silicon, FeSO4, and urea significantly increased POD activity compared to no foliar application by 33.45%, 30.60%, 23.13%, and 17.44%, respectively  treatment compared to the control treatment (28.9%)  and control (30.96 g) treatments  was observed in seed priming in combination with ZnSO4 foliar application. This was statistically similar to the foliar application of silicon  at seed priming treatment. Foliar application of ZnSO4 at seed primed and non-primed seed treatments increased GY by 18.51% and 9.60%, respectively. In addition, silicon foliar application at seed primed and non-primed seed treatments improved GY by 13.52% and 12.15%, respectively  and Zn (42.0 mg kg−1 DW) content in seed priming treatment was significantly higher than control. Maximum grain zinc content (49.4 mg kg−1 DW) was achieved in ZnSO4 foliar spray. In comparison to the control, foliar applications of ZnSO4 enhanced grain Zn content by 29.32%. Only FeSO4 foliar application significantly increased grain Fe content compared to no foliar application. Grain Fe content ranged from 51.1 mg kg−1 DW for no foliar application to 61.1 mg kg−1 DW for FeSO4 foliar application . Furthermore, there was a positive association among GY with BY , spike per m2 , kernels per spike , 1,000-grain weight , and Zn content in grains . A positive correlation was found between BY, spike per m2, kernels per spike, 1,000-grain weight, MSI, SOD and POD activity, Chl T, carotenoid, proline concentration, and grain Zn content. MSI was positively associated with Chl T , carotenoid , proline , GB , SOD activity , POD activity , grain Zn content , and grain Fe content , while MSI showed a strong negative relation with MDA content . The MSI is positively related to all traits except grain protein content while there is a significant negative relation between MDA and all traits investigated excluding grain protein content (no significant correlation between MDA with grain protein content). There was no significant relationship between Zn, Fe, and protein content of grains. Grain protein content exclusively had a significant association with proline and GB concentrations, and SOD and POD activity  increased photosynthetic pigments in both primed and unprimed seed treatments; however, foliage spraying had the greatest effect in primed seeds  and antioxidants that are not enzyme-based  . Seed prmed seed . In prevmed seed .Stress induced by water and nutrient deficiency results in oxidative stress in plants, causing membrane lipid peroxidation. Tissue MDA amounts represent membrane lipid peroxidation, which eventually damages integrity of membranes . The MSI4, silicon, and FeSO4 during the terminal stage significantly increased kernels per spike, 1000-grain weight, BY, and GY in comparison with the control  + FeSO4.7H2O (50 ppm) + ZnSO4.7H2O (50 ppm) + silicon (20 mg L−1) solution] and foliar application of these compounds  improved physiological and yield characteristics. Foliar application of ZnSO4, FeSO4, and urea drastically affected grain Zn, Fe, and protein content, respectively. To improve drought resistance, GY, and quality of rainfed wheat, seed priming and foliar spraying are recommended as crop management practices. Future investigations are necessary to assess the influence of seed priming and foliar spray of these compounds at various growth stages on rainfed wheat in various cultivars and soils.Results from this study have clearly demonstrated that the combination of seed priming [by urea (20 g LThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.LM: Formal Analysis, Project administration, Software, Validation, Writing – original draft. AS: Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – review & editing.


Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories.Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriously difficult to express in microbes. To improve their expression in The online version contains supplementary material available at 10.1186/s12934-023-02219-7. In planta, P450s unlock the diversity of secondary metabolism [Escherichia coli or Saccharomyces cerevisiae, allows the production of high-value natural products without the problems associated with the native hosts: seasonal nature of the production, low amounts of the product per plant, and tedious extraction from plant material. Well-known examples include the semi-synthetic production of the antimalarial drug artemisinin [de novo biosynthesis of analgesic opioids [Cytochromes P450 (CYPs or P450s) are amongst the most versatile enzymes in nature and draw the attention of metabolic engineers with their ability to catalyse unique reactions on a myriad of complex substrates. tabolism  and playtabolism , 3. Reco opioids , 7. StilE. coli – without eukaryotic organelles and no native P450 enzymes – a particularly challenging host. The N-terminal hydrophobic amino acids, which anchor P450s in membranes and act as a membrane localisation signal, cause the proteins to aggregate in inclusion bodies if the proteins cannot assume their native fold at a pace matching the speed of translation [E. coli [E. coli membrane proteins [All plant P450s require a redox partner for electron transfer and most localise on the membrane of the endoplasmic reticulum , 9, makinslation , 11. Larnslation . Barnes [E. coli . N-termi[E. coli –18. Beca[E. coli , 20, alt[E. coli  and exchproteins , 23 or wproteins .Although researchers can now choose from an arsenal of engineering strategies, no clear favourite exists and establishing novel plant pathways requires extensive optimisation and screening of the P450 enzyme expression and functionality , 23. ConE. coli cell factories by systematically comparing six common N-terminal modifications. We evaluate the changes of in vivo activity of CYP79A2 together with two members of the CYP83 family and then validate the best strategy on four additional P450 enzymes. CYP79 and CYP83 catalyse two consecutive steps at the entrance to the glucosinolate pathway and accept standard amino acids as substrates  [E. coli leader sequence from outer membrane protein A (OmpA) [We chose the conversion of phenylalanine to phenylacetaldoxime (Phe-Ox) catalysed by CYP79A2 for the initial assessment of N-terminal modifications because CYP79A2 constitutes a bottleneck in its biosynthetic pathways , 28, ande (SohB) , and (4)A (OmpA)   [To validate transmembrane domain truncation as a strategy that could be applied to a broad range of P450s, we chose four additional CYP79 enzymes with diverse N-terminal sequences, distinct substrate preferences and different plant origins. CYP79A1 from ng oxime , CYP79B2ubstrate , CYP79D2oleucine , and CYPalanine) . We clonm/z ratios and a characteristic fragmentation pattern in the mass spectrum [We assayed the enzymes by measuring the generation of putative glutathione conjugates derived from their respective amino acid substrates by tandem mass spectrometry (Q-TOF LC-MS/MS), comparing the relative production in strains expressing the native and truncated P450s  of USER overhangs (31 − 26 °C) with 5 min at each temperature step and finished with 30 min at 10 °C. The heterologous genes were assembled into synthetic operons with a maximum of three genes per operon. All operons were flanked by consensus T7 promoters with T7 terminators, and all genes shared the same ribosome binding site originating from the T7 bacteriophage major capsid protein.Purified PCR products were assembled using an adapted USER protocol from Geu-Flores et al.  and CavaE. coli (SohB), and (7) addition of E. coli outer membrane protein A (OmpA) signal peptide in front of the full-length P450 sequence (OmpA). 28aa, SohB and OmpA sequences were ordered from Twist Bioscience as short DNA fragments, amplified with uracil-containing primers, and used to create modified P450s on a separate plasmid backbone before they were cloned into the expression vector. Barnes sequence was short enough to be inserted into the primer overhangs that were used to create the modifications (3) and (4), and truncated P450s were prepared with primers annealing at within the gene sequences. DNA sequences of all used modifications are shown in Table To define transmembrane domains of P450 enzymes, we used Uniprot annotations in combination with a transmembrane domain prediction tool TMHMM v2.0 (now deprecated in favour of DeepTHMHH)  Table 11. To ide2PO4-K2HPO4 buffer pH 7.0, 0.05% glucose, 0.5% glycerol, 0.2% α-lactose, 2 mM MgSO4, and trace metal mix . To improve heme biosynthesis necessary for functional P450 enzymes, 0.5 mM 5-aminolevulinic acid was supplied to the media. Corresponding antibiotics were also added at the following concentrations: carbenicillin (50 µg/mL), spectinomycin (50 µg/mL), kanamycin (50 µg/mL) and chloramphenicol (34 µg/mL). 1 mM L-homophenylalanine  was supplemented to the strain expressing BvCYP79F6. Glutathione is one of the most abundant metabolites in E. coli [Overnight cultures were started by picking single colonies into 1.2 mL LB media with appropriate antibiotics in a 24-well plate. Then, the overnight cultures were diluted 1000-fold into 1.2 mL autoinducing medium in round bottom 24-well plates and the fermentation was carried out in a rotary shaker at 18 °C, 200 RPM for 72 h. Autoinducing medium, adapted from Studier et al. , was use E. coli  and as s2HPO4.7H2O, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 2 mM MgSO4, 1mM thiamine, 0.1 mM CaCl2, 0.5 mM 5-aminolevulinic acid, 0.05% glucose, 0.5% glycerol, 0.2% α-lactose and trace metal mix .M9 minimal media recipe was adjusted to allow for autoinduction of protein expression, and supplemented with 5-aminolevulinic acid, thiamine and trace metals. The final composition was 12.8 g/L Nag for 5 min. The media samples were stored at − 20 °C and the cell pellet at − 80 °C until analysis. For metabolite analysis an aliquot of the media was diluted 25-fold in water and filtered through 0.22 μm filters. The glutathione conjugates derived from Tyr, Trp, Leu, Val and HPhe were analysed with Q-TOF LC-MS/MS using their characteristic fragmentation pattern as described previously [13 C-,15 N-labeled amino acid mix . The titres were quantified with 13 C-, 15 N-Phe as internal standard using response factors calculated from dilution series of the analytes in spent E. coli media to account for the matrix effect  in RStun 4.2.2,  relying n 4.2.2,  and ggpln 4.2.2,  packagesn 4.2.2,  as previn 4.2.2, .Below is the link to the electronic supplementary material.Supplementary Material 1


N-docosahexaenoylethanolamine (DHEA), or synaptamide, is an endogenous metabolite of docosahexaenoic acid (DHA) that exhibits synaptogenic and neurogenic effects. In our previous studies, synaptamide administration inhibited the neuropathic pain-like behavior and reduced inflammation in the central nervous system following sciatic nerve injury. In the present study, we examine the effect of synaptamide on the peripheral nervous system in a neuropathic pain condition. The dynamics of ionized calcium-binding adapter molecule 1 (iba-1), CD68, CD163, myelin basic protein, and the production of interleukin 1β and 6 within the sciatic nerve, as well as the neuro-glial index and the activity of iba-1, CD163, glial fibrillary acidic protein (GFAP), neuronal NO synthase (nNOS), substance P (SP), activating transcription factor 3 (ATF3) in the dorsal root ganglia (DRG), are studied. According to our results, synaptamide treatment (4 mg/kg/day) (1) decreases the weight-bearing deficit after nerve trauma; (2) enhances the remyelination process in the sciatic nerve; (3) shows anti-inflammatory properties in the peripheral nervous system; (4) decreases the neuro-glial index and GFAP immunoreactivity in the DRG; (5) inhibits nNOS- and SP-ergic activity in the DRG, which might contribute to neuropathic pain attenuation. In general, the current study demonstrates the complex effect of synaptamide on nerve injury, which indicates its high potential for neuropathic pain management. Currently, one of the most urgent topics for research in neuroscience is chronic pain management, in particular, the management of chronic neuropathic pain that is caused by injury to the peripheral nervous system and leads to the development and maintenance of many somatic and psychophysiological disorders ,2.Gamma-aminobutyric acid (GABA) analogs and inhibitors and tricyclic antidepressants are among the first-line treatment options for neuropathic pain syndrome ,4, whereSome of the recently developed alternative methods for neuropathic pain treatment include substances of natural origin, such as polyunsaturated fatty acids (PUFAs) and their metabolites, that can act as neuromodulators and anti-inflammatory compounds in both peripheral and central nervous systems, prevent cytokine secretion, and block the synthesis of prostaglandins, leukotrienes, and thromboxanes ,7. In paSynaptamide is another endogenous metabolite of DHA that can promote neurite- and synaptogenesis in neuronal cell cultures ,16,17,18We have previously discovered that synaptamide treatment after the chronic constriction injury of the sciatic nerve affects the neuropathic pain development in rats, resulting in significantly less pronounced pain-like behavior ,25,26, iThus, in our current research, we investigate the effects of synaptamide treatment on (1) neuropathic pain severity by evaluating the weight-bearing difference in vehicle- and synaptamide-treated rats following chronic constriction injury (CCI) of the sciatic nerve; (2) the regeneration process by assessing the remyelination in the injured nerve and activating transcription factor 3 (ATF3) activity in the primary sensory neurons in the dorsal root ganglia (DRG) by immunohistochemical studies; (3) inflammation in the sciatic nerve and DRG by immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA); (4) satellite glial cells, neuronal NO-synthase (nNOS) and substance P (SP) activity in the DRG by immunohistochemical studies. We hypothesize that synaptamide treatment of neuropathic pain syndrome caused by sciatic nerve injury reduces inflammation in the peripheral nervous system, which results in downregulation of nNOS and SP activity, and thus, contributes to neuropathic pain attenuation.Vehicle-treated sham-operated rats (the “Sham” group) and synaptamide-treated sham-operated rats (the “Sham + Syn” group) symmetrically distributed their weight on their hind limbs throughout the experiment. At week 1 post-surgery, the weight distribution pattern was evaluated as 50.7 ± 1.4%:49.3 ± 1.4% between left and right limbs, respectively, in the “Sham” group and 49.1 ± 1.5%:50.9 ± 1.5% in the “Sham + Syn” group .However, 1-week post-surgery, the weight distribution pattern for vehicle-treated rats with CCI of the sciatic nerve (the “CCI” group) changed drastically: 63.9 ± 2.2%:36.1 ± 2.2% between intact and injured limbs, respectively . HoweverAt week 2, the asymmetry in weight distribution in the “CCI” group decreased to 55.0 ± 1.4%:45.0 ± 1.4%; whereas rats in the “CCI + Syn” group demonstrated almost symmetrical weight distribution between intact and injured limbs: 51.0 ± 1.4%:49.0 ± 1.4% . In the At week 3 and up to the end of the experiment, the weight distribution was almost equal in both the “CCI” and “CCI + Syn” groups . In partChronic constriction injury of the sciatic nerve was followed by swelling and derangement of the nerve fibers distal to the site of trauma , which cp < 0.001), and 6.1 ± 0.5% in the “CCI + Syn” group (p < 0.001) (p < 0.01) (p < 0.01) and “Sham + Syn” groups   B,E. Howe < 0.01) B,E. Neve< 0.001) B,E.Axonal regeneration in damaged nerves is accompanied by the inflammatory process, which involves the activation, proliferation, and migration of macrophages to the site of injury ,28. In tp < 0.001) and “Sham + Syn”  groups (p < 0.001)   is considered the morpho-chemical indicator of macrophage activity upregulation . Increas) groups A,D. No s) groups D. Howeve< 0.001) A,D. Neve + Syn”) A,D.p < 0.001) and “Sham + Syn”  groups (p < 0.05)   groups C,F. At t) groups F. Howeve < 0.05) C,F. At t + Syn”) C,F.p < 0.001) and “Sham + Syn”  groups (p < 0.01) (p < 0.001) and “Sham + Syn”  groups  groups B,E. Howe < 0.01) B,E. At d) groups B,E. Howe) groups E.p < 0.01) and “CCI + Syn” groups , compared to the “Sham” (100 ± 7.6%) and “Sham + Syn” (99.8 ± 5.5%) groups at day 14 post-surgery  and “Sham + Syn”  groups (p < 0.01) (p < 0.01)  groups D. Howeve < 0.01) D. The op < 0.01) D. Notabl < 0.01) D.p < 0.01) for small, 7.9 ± 0.2 (p < 0.001) for medium-sized, and 12.0 ± 0.3 (p < 0.001) for large neurons  form a distinct functional and morphological unit with each sensory neuron within the dorsal root ganglia. SGCs perform ion sink, insulation, and neurotransmitter recycling, and also buffer the extracellular environment via potassium and calcium channels. After peripheral nerve injury, SGCs undergo morphological and molecular alterations, including proliferation and an increase in coupling via gap junctions ,37. The  neurons D. Notabl neurons D. The nu neurons A,D. At t neurons A,D. p < 0.001), “Sham” , and “Sham + Syn”  groups  (p < 0.001) and “Sham + Syn”  (p < 0.001) and both sham-operated groups (p < 0.001) , “Sham + Syn” (2.7 ± 0.1), “CCI” (3.4 ± 0.2), and “CCI + Syn” (3.1 ± 0.2) E. The ne) groups B,E. Intectively) B,E. A si < 0.01) B,E. Howe< 0.001) B,E.p < 0.001) and “Sham + Syn” (p < 0.001) (p < 0.05) in the “CCI + Syn” group, in comparison to the “CCI” group (7.8 ± 0.8%)  C,F. No s< 0.001) F. Howeve ± 0.8%) C,F. GFAP ± 0.8%) C,F.p < 0.05) and “Sham + Syn”  groups (p < 0.001) and “Sham + Syn”  groups  and “CCI + Syn” (4.6 ± 0.4%) groups in comparison to the “Sham”  groups A,C. At d) groups A,C.p < 0.001) (p < 0.05) (p < 0.01), “Sham + Syn” (p < 0.05), and “CCI + Syn” (p < 0.05) groups (Synaptamide treatment after the chronic constriction injury resulted in a significant increase in the immunoreactivity of the CD163-positive microglia/macrophages in both “CCI + Syn” (1.3 ± 0.1%) and “Sham + Syn” (1.5 ± 0.1%) groups 14 days post-surgery B,D. CD16< 0.001) D. Also,  < 0.05) B,D. Inte < 0.05) D. Howeve < 0.05) D. The CD) groups B,D.3 in the “Sham” group and 2595 ± 295 cells per mm3 in the “Sham + Syn” group  and “Sham + Syn” (p < 0.001) (3 in the “Sham” group and 2027 ± 200 cells per mm3 in the “Sham + Syn” group (3 in vehicle-treated rats in comparison to the “Sham” (p < 0.01) and “Sham + Syn” (p < 0.001) groups   A,D. At dn” group A,D. Chro) groups A,D. Howe < 0.05) A,D.3 in the “Sham” group and 4921 ± 302 cells per mm3 in the “Sham + Syn” group (3 in comparison to “Sham” (p < 0.05) and “Sham + Syn” (p < 0.05) (3 in the “CCI + Syn” group at 14 days after surgery (3), in comparison to the “CCI” group  (3) and “Sham + Syn” (4081 ± 218 cells per mm3) groups  is considered to be one of the fundamental causes of neuropathic pain . At day n” group E. Periph < 0.05) B,E. Foll surgery B,E. Howe surgery E. Nevert< 0.001) B,E. Nota) groups B,E.3), in comparison to the “Sham”  and “Sham + Syn”  groups  (p < 0.01), 14 days post-surgery (3) and “CCI + Syn” (3767 ± 410 cells per mm3) groups, in comparison to the “Sham”  and “Sham + Syn”  groups  groups C,F. Howe < 0.05) C,F. Neve-surgery F. At day) groups C,F. Howe) groups F.Neuropathic pain treatment options include several recently developed alternative methods, such as using substances of natural origin, in particular, the polyunsaturated fatty acids (PUFAs) and their metabolites, which can act as neuromodulators and anti-inflammatory compounds in both peripheral and central nervous systems ,7. DocosPeripheral nerve injury results in the development of neuropathic pain syndrome involving spontaneous ongoing pain ,2, the sSciatic nerve injury is known to induce cellular and molecular changes in the peripheral nervous system, which includes the distal and proximal segments of the nerve and the dorsal root ganglia. Chronic constriction injury leads to dramatic changes to the sciatic nerve, involving demyelination and degradation of myelin sheaths, recruitment of inflammatory cells to the site of the trauma, and derangement of nerve fibers , all of In our previous study, we have shown that synaptamide treatment after CCI reduces inflammation in the lumbar spinal cord by downregulating iba-1-positive microglia activity, as well as IL1β concentration 35 days post-surgery . Iba-1  receptors on the cellular membranes of the astrocytes [In addition, synaptamide treatment affects microglia/macrophages, satellite glial cells (SGCs), and neurotransmitter systems in the DRG after the CCI. It is known that each sensory neuron within the dorsal root ganglia forms a distinct functional and morphological unit with its SGCs. Satellite glial cells possess a number of similarities to astrocytes in the CNS; they share common functions such as ion sink, insulation, and neurotransmitter recycling, as well as some immunocytochemical properties, apart from the expression of the glial fibrillary acidic protein. GFAP is the basic protein of astrocytes’ intermediate filaments; an increase in its expression level is considered to be a morpho-chemical indicator of astroglial activation in the CNS , since a control . Supposetrocytes .In addition to the pronounced activity of synaptamide regarding satellite glial cells, synaptamide treatment significantly decreased the number of nNOS- and SP-positive neurons in the DRG at day 35 post-surgery. Substance P and NO are the most important mediators of neuropathic pain and are involved in the manifestation of allodynia and hyperalgesia, which are typical for neuropathic pain syndrome ,60,61. IIn general, the data collected in the current study show that synaptamide has a complex effect on the post-injury processes in the peripheral nervous system. Considering the anti-inflammatory properties of synaptamide in the central nervous system shown in our previous studies ,24,25,26This study presents some limitations. The effect of synaptamide administration during the acute phase of sciatic nerve chronic constriction injury has not yet been examined. The study was carried out on male rats only; the effect of synaptamide administration in neuropathic pain conditions on female specimens concerning menstrual cycles has yet to be studied.Berryteuthis magister was used to obtain the concentrate of PUFAs, which was later converted into ethyl esters and treated with ethanolamine . Then, high-performance liquid chromatography on a preparative reverse-phase column Supelco Discovery HS C-18 , 10 µm particle size, 250 mm × 50 mm i.d., was performed on a Shimadzu LC-8A chromatograph  with UV/VIS SPD-20A (205 nm), using the isocratic elution with 50 mL/min rate and the system ethanol/water , to separate fractions containing synaptamide from the ethanolamines concentrate. Synaptamide fractions were then analyzed by gas chromatography and gas chromatography–mass spectrometry after evaporation under a vacuum.The concentrate of N-docosahexaenoylethanolamine  B with a The experiments involved male Wistar rats  that were raised at the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia. The rats were housed 3–4 per cage at a constant temperature (23 ± 2 °C) and humidity (55 ± 15%) on a daily 12-h light/dark cycle, with free access to food and water. All procedures were approved by the Animal Ethics Committee at the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences (No. 3-221019 from 22 October 2019), according to the international regulations of the European Directive 2010/63/EU and ethical guidelines for the study of experimental pain in conscious animals by the International Association for the Study of Pain.Chronic constriction injury of the sciatic nerve was used as a model of neuropathic pain, as originally described by Bennett and Xie . Before The severity of pain in the injured hind limb was evaluated by the weight-bearing difference test, as previously described by Nakazato-Imasato and Kurebayashi . BrieflyExperimental rats were sacrificed on day 14 (n = 56) or 35 (n = 56) after the chronic constriction injury C. The suAfter processing and embedding in paraffin, the 7 μm slices of the samples were prepared on a Leica rotary microtome RM 2245 . For the histological examination, following deparaffinization, the longitudinal slices of the sciatic nerve were stained with hematoxylin–eosin, and the DRG slices were stained with toluidine blue . The slices were then dehydrated and embedded in a mounting medium  for further examination.2O2 in 0.1 M phosphate buffer (pH 7.2), and the non-specific binding of antibodies was blocked by a 60 min-long incubation in a 2% bovine serum albumin solution  with 0.25% Triton X-100 . Subsequently, the sections were incubated with primary antibodies for 24 h at 4 °C. In particular, the sections of DRG were stained with mouse monoclonal antibodies to GFAP ; iba-1 ; substance P ; nNOS , CD163 ; and ATF3 . The sections of the sciatic nerve were incubated with mouse monoclonal antibodies to MBP ; rabbit mono- and polyclonal antibodies to iba-1 ; CD163 ; CD68 . Negative control with no primary antibodies was also performed. Three portions of 0.1 M phosphate buffer (pH 7.2) were used to wash the slices after primary antibodies. Each section was then treated with appropriate secondary antibodies labeled with horseradish peroxidase  for 30 min. Following three washes in 0.1 M phosphate buffer (pH 7.2), the slices were stained with chromogen  for 10 min to elicit the immunoperoxidase reaction. After washing and dehydrating, the slices were embedded in a mounting medium  for further examination.For the immunohistochemical studies, the longitudinal slices of the sciatic nerve , the transverse slices of the sciatic nerve (for MBP staining), and the slices of DRG were deparaffinized and incubated in a 3% hydrogen peroxide solution to block endogenous peroxidase activity. The slices were then washed from H3 was then calculated using the formula:3; n—the number of cells within the area of interest on a section; S—the area of interest on a section in µm2; 7—the thickness of the slices in µm; 1,000,000,000—conversion factor for µm3 and mm3.For the image analysis, photographs of the slices after histological and immunohistochemical staining (at least 70 per group) were captured with a CCD camera, AxioCam HRc , on a microscope, Axio Image Z2 . On each photograph, immunoreactivity in the DRG and sciatic nerve was assessed using ImageJ software . Neuro-glial index on the toluidine blue-stained slices of the DRG was counted using the Cell Counter plugin. The Cell Counter plugin was also used to calculate the number of nNOS-, SP-, and ATF3-positive cells per section within the area of interest in the DRG and the number of CD163-positive cells per section within the area of interest in the sciatic nerve. The number of cells per mmThe IHC Toolbox plugin was used to evaluate the area fraction of the iba-1-, MBP-, and CD68-positive staining areas in the sciatic nerve slices and the GFAP-, CD163-, and iba-1-positive staining areas in the DRG. For the staining area evaluation, visually selected pixels were used for the statistical color detection model, which was later used for the background elimination. After binarization, the area of interest was selected for each photograph, and the area fraction was measured and expressed as the percentage of the colored area on the total area of interest.The concentration of IL-1β and IL-6 in the samples of sciatic nerves of the experimental rats  was determined by ELISA. First, the extraction buffer , containing 1 mg/mL of protease inhibitor cocktail  and 0.01 mg/mL of phosphatase inhibitor cocktail  on ice, was used for homogenization of the samples. The rat ELISA kits for IL-1β  and IL-6  were used to evaluate the cytokine concentrations in the sciatic nerve samples. A BCA protein assay kit  was then used to assess the total protein concentrations. iMark microplate absorbance reader  was used to measure light absorbance at 450 nm wavelength.p < 0.05 was taken as statistically significant. The datasets obtained from ELISA , toluidine blue, and the immunohistochemical studies  were compared using one-way ANOVA tests followed by a post hoc Tukey’s multiple comparison test. All data were analyzed using GraphPad Prism 4  and shown as mean ± SEM.The datasets obtained from the weight-bearing test  were subjected to statistical analysis with the Mann–Whitney test and