CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6–9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley β-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G4) and cellppentaose (G5) to cellobiose (G2) and cellotriose (G3). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications.Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-β-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-β-1,4-glucanase gene ( Cellulose is the principal component of plant lignocellulose; it is a polysaccharide composed of glucose linked by β-1,4-bonds . OligosaIn industrial applications, enzyme production is the most critical and costly step. In nature, numerous microorganisms produce endonucleases, such as fungi, bacteria, and actinomycetes . MyxobacMyxococcus sp. B6-1 from the soil. In this study, a novel endo-β-1,4-glucanase gene from Myxococcus sp. B6-1 was cloned and expressed in E. coli. We further investigated the biochemical characteristics and straw saccharification ability of recombinant enzymes. To the best of our knowledge, this is the first report of GH6 cellulase from Myxococcus sp.We previously isolated and identified a strain of 2-G5), carboxymethyl-cellulose (CMC), barley β-glucan, laminarin, xylan (from corncob), avicel, chitosan, and filter paper were obtained from McLean Biochemical Technology Co., Ltd. , whereas lichenin was purchased from Megazyme . Other chemicals used in this study were provided by Sigma-Aldrich .Glucose and other cello-oligosaccharide (GMyxococcus sp. B6-1 was isolated from soil and cultured in a lysogeny broth (LB). DH5 and BL21 (DE3) strains of E. coli from our laboratory were respectively utilized for gene cloning and expression. Plasmid pET-29a  was used for expression.Myxococcus sp. B6-1 and extracted using a previously described method [CelA257 (GenBank registration number: MW248127) was amplified with the primers: Cel257F (5′- TAAGAAGGAGATATACATATGTGGCTCGCGGCCTCG-3′) and Cel257R (5′-GTGGTGGTGGTGGTGCTCGAGCGTACCGTCGATGAG-3′). PCR program included denaturation at 98 °C for 2 min, followed by 30 cycles: denaturation at 98 °C for 10 s, annealing at 65 °C for 5 s, polymerization at 72 °C for 10 s, with a final extension at 72 °C for 10 min. PCR products were purified, digested with NdeI and XhoI, and ligated into a pET-29a(+) vector to form pET-29a-CelA257. Following plasmid transformation into competent E. coli DH5α, positive transformants were isolated and sequenced. A verified recombinant plasmid was cultured on a LB medium containing kanamycin 50 mg/mL at 37 °C in E. coli BL21 (DE3). Isopropyl-β-D-thiogalactopyranoside (IPTG) solution (0.2 mM) was induced for protein overexpression until OD 600 of strain reached 0.5–0.6. Following that, recombinant strain cells were gathered and lysed after being centrifuged at 8000× g for 20 min. After ultrasonic fragmentation , centrifugation was performed at 12,000 r/min and 4 °C for 15 min. The supernatant of recombinant CelA257 with a 6-His tag was purified with a Ni-NTA column and eluted with 20 mM Tris-HCl (pH 7.0) containing various imidazole concentrations (50–400 mM). Bradford’s method was used to determine the protein concentration [The DNA of genomic organisms was isolated from d method . The endhttps://web.expasy.org, accessed on 23 July 2020). The protein sequence was analyzed using the NCBI database , and the sequence of amino acids analysis of CelA257 to other sequences was conducted by Clustal omega tools. Domains of structural and active sites were predicted using the Pfam database. Mega 5.0 Software and the Poisson model adjacency method were used to construct the phylogenetic tree. In addition, a 3D protein structural model of CelA257 was generated by the alpha fold2 online server , and the structure of the active site was generated using Cel6 from Mycobacterium tuberculosis (PDB: 1UP3) as a template. Cel6 shared 42% identity and 60% query cover with the CelA257 sequence. Molecular graphics software PyMol was used to visualize the crystal 3D structure model.Calculations were performed to determine the molecular mass and the isoelectric point of the compound using the Expasy tool (w/v) was dissolved in 20 mM sodium phosphate buffer (pH 6.0) and incubated with enzyme for 1 h at 50 °C to determine enzyme activity The reducing sugar was determined according to the dinitrosalicylic acid (DNS) method [w/v) barley β-glucan, lichenan, PASC, laminarin, avicel, filter paper, xylan, and chitosan were utilized. Triplicates of all experiments were performed. Chitosan substrate was prepared as previously described [CMC  method . For theescribed . The amoCelA257 was tested in different buffers (with pH 3.0–10.5) to determine its optimal pH, including citrate buffer , sodium phosphate buffer , tris-HCl buffer , and glycine-NaOH buffer . Various buffers (pH 3.0–10.5) were incubated at 4 °C for 24 h with aliquots of recombinant CelA257 in order to determine pH stability. The optimal reaction temperature was identified using a wide range of temperatures (4–100 °C) in pH 6.5 sodium phosphate buffer for 1 h. CelA257′s thermal stability was analyzed by depositing the solution in sodium phosphate buffer (pH 6.5) at 30–60 °C for 48 h. The impact of various metal ions (1 mM) and chemicals was measured in sodium phosphate buffer (pH 6.5). Reactants without any metal ions and chemicals were employed as controls, and the enzyme activity was considered 100%. To obtain the enzyme’s kinetic parameters, enzyme activity was determined using various CMC concentrations (1.2–4.0 mg/mL) in phosphate buffer (pH 6.5) at 50 °C for 1 h. The data were graphed using the Lineweaver–Burk method.2-G5) in sodium phosphate buffer (pH 6.5) at 50 °C with 0.1 U CelA257. The samples were withdrawn after 6 and 12 h and boiled separately for 10 min. Following that, an analysis of the end products was conducted by thin layer liquid chromatography method (TLC). The final products of the reaction were applied onto silicone glass plates from Merck, Germany. These plates were then treated with a solvent system consisting of n-butanol, water, and acetic acid in a 2:1:1 v/v/v ratio. The solution was visualized by spraying with sulfuric/methanol acid  and heating at 85 °C for 15 min.The hydrolysis mode of CelA257 was confirmed by hydrolyzing 1 mg/mL cello-oligosaccharides (Gw/v) were crushed through a 40-mesh sieve followed by blending in 20 mM PBS buffer (pH 6.5) containing 0 or 0.45 U crude CelA257 enzyme. Then the mixture was transferred to a 10 mL centrifuge tube, shaken at 50 °C for 48 h, and then boiled for 10 min, followed by centrifugation. The supernatants were utilized to determine the released reducing sugars of the reaction product using the DNS method. Finally, the number of milligrams of the reducing sugar released per milliliter of the reaction system was calculated.Different agricultural straws at 1%  of 1404 bp length that encoded 467 amino acids with the first 11 amino acids as a signal peptide sequence. Therefore, CelA257 must be an extracellular enzyme. The signal peptide was removed during heterologous expression. The theoretical pI was 5.01, and the predicted molecular mass was 48.7 kDa. Additionally, the phylogenic tree . BLAST analysis manifested that CelA257 shared 54.7% of its identity with CelA1 from Streptomyces halstedii [Streptomyces sp. KSM-9  [Cellulomonas fimi  [Thermobifida fusca  [Sequence alignment analysis of endo-β-1,4-glucanase gene nic tree a demonstalstedii , followe13933.3) , CenA fr07984.1) , E2 from26222.2) , and sha26222.2) . These eMycobacterium tuberculosis [CelA257 was predicted using the Pfam database to have structural domains from the CBM4-9 and GH6 families b. The amN1) from Cellulomonas fimi and comprised a typical jelly-roll-sandwich with two antiparallel β-sheets [Protein 3D modeling results revealed a narrow tunnel in the center of CelA257, where the active site was located . CelA257β-sheets .CelA257 gene was successfully expressed in E. coli. SDS-PAGE analysis of recombinant CelA257 soluble expression induced by IPTG was shown in the supernatant  methanol, acetonitrile, isopropanol, ethanol, 20 mg/mL tritonX-100, tween 80, 10 mM EDTA, and 2 mg/mL SDS. The residual activity remained more than 80% when 10% acetone and 5% DMSO were added.CelA257 activity was raised by 1 mM Coectively . It was  library . Cu2+, Znhibited . TherefoCelA257 was relatively highly active against CMC (3.64 U/mg), barley β-glucan (17.58 U/mg), lichenan (6.49 U/mg), PASC 2.42 U/mg), and avicel (2.82 U/mg), but was inactive against chitosan (0.72 U/mg), laminarin (0.78 U/mg), and filter paper (0.54 U/mg) . The res U/mg, anmK and maxV values were 3.24 mg/mL and 4.74 U/mg toward CMC, respectively. mK value was higher than Cel6H-p35 (1.31 mg/mL) and Cel6H-p23 (1.32 mg/mL), but maxV value was lower than Cel6H-p35 (8.75 U/mg) and Cel6H-p23 (6.57 U/mg) [mK value indicated that the affinity between the enzyme and the substrate was reduced, and the enzyme was not conducive to hydrolysis of the adjacent β-1,4-glucan linkages, resulting in the decrease in the maxV values. The results suggested that CBM4-9 probably improved the binding to crystalline cellulose but not soluble cellulose, such as CMC.The enzyme kinetic parameter was analyzed by Lineweaver–Burk plots see . Km and 57 U/mg) . The hig4 and G5 rapidly at 6 h but did not hydrolyze G2 and G3 under reaction conditions. CelA257 hydrolyzed both G4 and G5 to G2 and G3. The results were similar to Cel6H-p35 from a compost metagenomic library. When Cel6H-p35 and CMC reacted for 12 or 24 h, Cel6H-p35 hydrolyzed cello-oligosaccharide G5 to G2 and G3 and G4 to G2 but hardly hydrolyzed G3 [The model of CelA257 was analyzed by degrading cello-oligosaccharides, and the hydrolyzed products were analyzed by TLC . CelA257lyzed G3 . It was Five kinds of agricultural straw powers were used to analyze the CelA257′s saccharification effect. Compared with the control, the reducing sugars produced by treatment groups with CelA257 were significantly increased after 48 h. CelA257 improved the enzymolysis efficiency of all agricultural straws. The enhanced reducing sugar concentrations in wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL) are shown in CelA257) was cloned and expressed in E. coli. CelA257 was stable at temperatures ranging from 4 to 50 °C and pH ranging from 6 to 9.5. The results of oligosaccharide hydrolysis indicated that CelA257 is an endo-type cellulase. CelA257 exerted broad substrate specificity and high activity to crystalline cellulose. CelA257 was added to agricultural straws to increase their saccharification efficiency. These findings suggested that it may be used as a biomass saccharification supplement.In this study, a novel endo-β-1,4-glucanase gene (


This activity was reviewed by CDC and was conducted consistent withapplicable federal law and CDC policy.***Daily mean PM2.5 was highest on June 7 for allregions, except the Adirondacks,µg/m3 (Western) to 122.3µg/m3 (NYC metro), representing 590% and1,229% increases, respectively, above 10-year baseline concentrations (8.0µg/m3 in Western and 9.2µg/m3 in NYC metro). During June 1–14,a total of 1,310 asthma-associated ED visits were identified through ESSS (2.5 and asthma-associated ED visit ρ ranged from0.31 (Western) to 0.80 . The largest region-specific increases inasthma-associated ED visits and highest ρ estimates were in the Eastern LakeOntario , Central , and Upper Hudson Valley  regions . Thesestrategies include collaboration with physicians to ensure proactive communicationabout the risks of wildfire smoke to their patients with asthma and with schools toensure effective wildfire smoke response plans.As wildfire smoke events become more frequent and widespread, the findings from thisanalysis can enhance risk communication and better focus response efforts towardpersons at increased risk for asthma exacerbations (


Cu(II) complexes were prepared by reaction of each benzoylhydrazone with Cu(II) acetate. All compounds were characterized by elemental analysis and mass spectrometry as well as by FTIR, UV-visible absorption, NMR or electron paramagnetic resonance spectroscopies. Complexes isolated in the solid state (1–8) are either formulated as [Cu(HL)acetate] (with L1 and L4) or as [Cu(Ln)]3 . Single crystal X-ray diffraction studies were done for L5 and [Cu(L5)]3, confirming the trinuclear formulation of several complexes. Proton dissociation constants, lipophilicity and solubility were determined for all free ligands by UV-Vis spectrophotometry in 30% (v/v) DMSO/H2O. Formation constants were determined for [Cu(LH)], [Cu(L)] and [Cu(LH−1)] for L = L1, L5 and L6, and also [Cu(LH−2)] for L = L6, and binding modes are proposed, [Cu(L)] predominating at physiological pH. The redox properties of complexes formed with L1, L5 and L6 are investigated by cyclic voltammetry; the formal redox potentials fall in the range of +377 to +395 mV vs. NHE. The binding of the Cu(II)-complexes to bovine serum albumin was evaluated by fluorescence spectroscopy, showing moderate-to-strong interaction and suggesting formation of a ground state complex. The interaction of L1, L3, L5 and L7, and of the corresponding complexes with calf thymus DNA was evaluated by thermal denaturation. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. The complexes show higher activity than the corresponding free ligand, and most complexes are more active than cisplatin. Compounds 1, 3, 5, and 8 were selected for additional studies: while these complexes induce reactive oxygen species and double-strand breaks in both cancer cells, their ability to induce cell-death by apoptosis varies. Within the set of compounds tested, 8 emerges as the most promising one, presenting low IC50 values, and high induction of oxidative stress and DNA damage, which eventually lead to high rates of apoptosis.We report the synthesis and characterization of a group of benzoylhydrazones (L Cancer is a leading cause of death in developed countries, with a significant expense to human lives. Despite the success of platinum metallodrugs, drawbacks such as intrinsic and acquired chemoresistance, as well as unspecific action leading to toxic side effects, drive researchers towards the search for alternative and safer chemotherapeutics, which may also take advantage of their different mechanisms of action. One alternative is the use of metallodrugs in which distinct metal ions may be explored to overcome these drawbacks and take advantage of different reactivity, electronic features and redox chemistry.Copper is a signalling metal ion involved in cell growth and proliferation. The field of cuproplasia (regulated copper-dependent cell proliferation) has just begun to be explored, but has already given proof of its importance in fighting cancer . It is kDespite being an essential nutrient to humans, copper homeostasis must be strictly maintained. Under biological conditions, copper is mostly bound to peptides and proteins, preventing uncontrolled redox activity. Its deficiency is detrimental, but its excess results in increased cellular oxidative stress. Organic molecules which act as chelating moieties can bind and sequester metal ions, or can act as ionophores that cross the cell membrane when still bound to the metal ion and release it inside the cell . In the 50 values from 1.4 nM to 32.13 μM (8-Hydroxyquinoline (8HQ) and its derivatives are versatile compounds and have been explored in the design of various classes of anticancer compounds, acting through different mechanisms of action . Numerou32.13 μM .in vitro and in vivo  complexes with 8HQ derivatives and their antiproliferative effect in breast cancer cells , increasing the ROS concentration in the cells, and triggering cell defences that lead to cell death.Recently, we reported on a series of six 8HQ-benzohydrazones and their coordination to V(IV)O ions . CytotoxDetails on the experimental part are included in p-toluic hydrazide (99%), and 4-aminobenzoic hydrazide (95%) were all from Aldrich, while 4-hydroxybenzhydrazide (puriss) was from Merck and isoniazid (≥99%) was from Fluka. [Cu(AcO)2]·H2O from Panreac was used as received. Dioxane (Fluka), dimethyl sulfoxide , methanol  and ethanol  were all analytical grade and used without further purification. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tetrabutylammonium nitrate (TBAN), glutathione and ascorbic acid were purchased from Sigma-Aldrich in puriss quality. KCl, HCl, KOH, DMSO, EDTA and potassium hydrogenphtalate were obtained from Molar Chemicals (Hungary) and used without further purification. Milli-Q ultra-pure water was used for the preparation of all solutions. For the reactions with AA and GSH, the pH of the solutions was adjusted to 7.4 with HEPES (50 mM). CuCl2 stock solution was prepared by the dissolution of CuCl2 in water and the concentration was determined by complexometry. When necessary, the pH was set to the appropriate value by the addition of HCl or KOH solutions. Elemental analysis for C, H and N, were carried out on a FISONS EA 1108 CHNS-O apparatus at Laboratório de Análises of Instituto Superior Técnico, while ESI-MS spectra of methanolic solutions of the compounds in both positive and negative modes were measured in a 500-M Varian Ion Trap Mass Spectrometer. Proton, carbon and correlation NMR spectra were obtained on a Bruker Avance II + 300 (UltraShieldTM Magnet) spectrometer operating at 300 MHz for proton and at 75.4 MHz for carbon, at room temperature; the chemical shifts are reported in ppm using tetramethylsilane as the internal reference. A JASCO FT/IR 4100 spectrophotometer was used for recording the infra-red spectra and electronic absorption spectra (UV-Vis) were recorded ether with a Perkin Elmer Lambda 35 (in the characterization studies) or an Agilent Cary 8,454 diode array spectrophotometer (in the spectrophotometric speciation studies). UV-vis spectrophotometers were thermostated to within ±0.1°C by Haake water baths. EPR spectra were recorded at 120 K with an X-band (9.4 GHz) Bruker EMX spectrometer equipped with an HP 53150 A microwave frequency counter with the following instrumental settings: microwave frequency, 9.40–9.41 GHz; microwave power, 20 mW; time constant, 163.8 m; modulation frequency, 100 kHz; modulation amplitude, 4 G; sweep time, 335.5 s; resolution 4,096 points. Magnetization measurements as a function of temperature were performed using a SQUID magnetometer (Quantum Design MPMS). The molar magnetic susceptibility values were subtracted for diamagnetism of the constituent atoms, estimated from Pascal constants, to isolate the copper spin contribution; the corrected molar susceptibility is designated by χm. Software PHI , 4-chlorobenzhydrazide (98%), 4-fluorobenzoic hydrazide (96%), 4-methoxybenzhydrazide (97%), ware PHI  was used1-L6 were described in a previous publication  were obtained from an acetone solution of the compound kept at room temperature for a few weeks.Crystals of L7: Bright yellow solid. Yield: 62.3%. Elem. analysis for C17H14N4O2·0.25H2O [found ]: C, 65.5% (65.69%); H, 4.5% (4.70%); N, 18.0% (18.03%). ESI-MS m/z (−) 305.40  (+) 306.99 . FTIR  3,441–3,318 (OH and primary amine), 3,212 (br), 3,109–3,046 (aromatic CH), 1,645 (carbonyl), 1,625 (CNquinoline), 1,548 (imine). UV-Vis  265 (2.2 × 104), 284 (2.8 × 104), 295 (2.9 × 104), 350 (2.4 × 104).L8: Bright yellow solid. Yield: 71.1%. Elem. analysis for C16H12N4O2·0.25H2O [found ]: C, 64.8% (64.75%); H, 4.2% (4.24%); N, 18.8% (18.88%). ESI-MS m/z (−) 291.21  (+) 293.13 . FTIR  3,445, 3,326, 3,046 (aromatic CH), 2,860 (azomethine CH), 1,679 (carbonyl), 1,549 (imine). UV-Vis  260 (2.0 × 104), 295 (2.35 × 104), 344 (1.3 × 104), 356 (1.37 × 104), 400 (1.5 × 103).Lca. 10 ml of MeOH. A few drops of 0.1 M KOH solution in MeOH were added till pH ≈ 9. In a vial, 0.25 mmol of [Cu(AcO)2]·H2O were dissolved in MeOH and added to the ligand precursors’ solution. The mixture was stirred for ca. 4 h at room temperature. In general, the ligand’s suspensions presented a yellow-milky aspect and, upon the addition of the blue copper solution, the reaction mixture gained a darker colour with the formation of a reddish-brown solid. The solids were collected by filtration, washed with ice-cold MeOH and dried under vacuum in a desiccator over silica-gel.[Cu(HL1)(AcO)] (1): Red solid. Yield: 85.7%. Elem. analysis for C19H15N3O4Cu∙H2O [found ]: C, 52.8% (52.96%); H, 3.5% (3.98%); N, 9.7% (9.75%). ESI-MS m/z (−) 410.96 . FTIR  3,415 (br), 3,054, 1,664 (br), 1,586, 1,512, 1,491, 1,454. UV-Vis  277 (2.18 × 104), 331 (2.3 × 104), 390 (1.2 × 104), 478 (2.4 × 103).[Cu(L2)]3 (2): Red solid. Yield: 95.3%. Elem. analysis for 3×{C17H10N3O2ClCu∙0.5H2O∙0.25MeOH} [found ]: C, 51.0% (51.25%); H, 2.7% (2.99%); N, 10.1% (10.39%). ESI-MS m/z (−) 385.23 . FTIR  3,428 (br), 3,021, 1,662 (br), 1,593, 1,506, 1,487, 1,446. UV-Vis  282 , 332 (8.0 × 103), 405 (6.0 × 103), 428 (5.5 × 103), 556 (3.8 × 103).[Cu(L3)]3 (3): Dark red solid. Yield: 66.2%. Elem. analysis for 3×{C17H10N3O2FCu∙1.25H2O} [found ]: C, 52.1% (51.91%); H, 2.8% (3.20%); N, 10.5% (10.68%). ESI-MS m/z (−) 369.39 ; m/z (+) 371.08 . FTIR  3,423 (br), 3,020, 1,662 (br), 1,601, 1,509, 1,493, 1,446. UV-Vis  280 , 327 (1.25 × 104), 394 (7.0 × 103), 425 (4.5 × 103), 535 (1.8 × 103).[Cu(HL4)(AcO)] (4): Reddish-brown solid. Yield: 74.9%. Elem. analysis for C20H17N3O4Cu∙0.5H2O [found ]: C, 54.9% (55.11%); H, 4.0% (4.16%); N, 9.6% (9.64%). ESI-MS m/z (+) 367.10 . FTIR  3,420 (br), 3,052, 1,662 (br), 1,608, 1,518, 1,496, 1,452. UV-Vis  278 (2.35 × 104), 327 (2.0 × 104), 402 (1.25 × 104), 426 , 540 .[Cu(L5)]3 (5): Reddish-brown solid. Yield: 39.9%. Elem. analysis for 3×{C18H13N3O3Cu∙1.75H2O} [found ]: C, 52.1% (52.17%); H, 3.6% (4.01%); N, 9.7% (10.14%). ESI-MS m/z (+) 383.08 . FTIR  3,386 (br), 3,046, 1,657 (br), 1,603, 1,506, 1,485, 1,449. UV-Vis  281 (1.45 × 104), 335 (1.5 × 104), 399 (9.0 × 103), 429 , 560 . Crystals suitable for SCXRD were obtained after a few weeks from the mother liquor (methanol).[Cu(L6)]3 (6): Brown solid. Yield: 62.0%. Elem. analysis for 3×{C17H11N3O3Cu∙1.5H2O} [found ]: C, 51.7% (51.58%); H, 3.2% (3.56%); N, 10.5% (10.61%). ESI-MS m/z (+) 367.23 . FTIR  3,414 (br), 3,060, 1,648 (br), 1,605, 1,524, 1,496, 1,454. UV-Vis  281 (2.25 × 104), 332 (1.9 × 104), 407 (1.2 × 104), 478 , 549 .[Cu(L7)]3 (7): Brown solid. Yield: 59.0%. Elem. analysis for 3×{C17H12N4O2Cu∙2.5MeOH} [found ]: C, 50.0% (50.06%); H, 3.9% (4.32%); N, 13.2% (13.34%). ESI-MS m/z (+) 399.51 . FTIR  3,440 (br), 3,342, 3,216, 3,036, 1,625, 1,602, 1,523, 1,493, 1,454. UV-Vis  291 (2.9 × 104), 345 , 417 (1.3 × 104), 535 .[Cu(L8)]3 (8): Reddish-brown solid. Yield: 52.7%. Elem. analysis for 3×{C16H10N4O2Cu∙1.75H2O∙0.25MeOH} [found ]: C, 49.5% (49.62%); H, 3.3% (3.72%); N, 14.0% (14.24%). ESI-MS m/z (+) 354.08 . FTIR  3,435 (br), 3,047, 1,628, 1,595, 1,521, 1,499, 1,460. UV-Vis  276 (1.1 × 104), 340 (9.0 × 103), 387 (6.0 × 103), 495 .In a round-bottom flask, 0.25 mmol of the corresponding ligand precursor were suspended in Ka values) of L1 - L6, have already been included in a previous publication in which their binding to V(IV)O was also disclosed . The synthesis, solid state and solution characterization  and L4 (methyl substituted) an acetate anion from the original salt seems to be maintained in the solid-state complex, in all other complexes double deprotonation of the ligand is assumed. Regarding the substituent effects, unsubstituted or substituents with inductive charge effect, like CH3, do not seem to be enough to doubly deprotonate the ligand. The other substituents, having a resonance effect on the aromatic ring, allow for the double deprotonation of the ligand and the coordination to the Cu(II) centre in a di-anionic form. All complexes were spectroscopically characterized, however, and due to the large number of compounds synthesized, with some techniques only selected compounds will be evaluated.Copper complexes are all new and were synthesized in moderate to good yields from the Cu(II) acetate salt. Elemental analysis and mass spectrometry reveal a 1:1 ligand-to-metal stoichiometric ratio for all. Interestingly, while with L−1, assigned to OH and NH stretching vibrations. A set of bands between 1,680 and 1,500 cm−1, due to vibrations of double bonds in the carbonyl and imine moieties, as well as in the aromatic rings, is observed. Coordination to copper leads to the disappearance of the sharp OH and NH bands, with a broad band appearing in the same region, probably associated with hydrogen-bonded OH and NH groups and solvent molecules retained in the solid matrixes  5 and its Cu-complex 5 were obtained from acetone and methanol, respectively. L5 crystallized in the triclinic system, space group P−1, and the asymmetric unit consists of two molecules of L5 and three water molecules engaged in hydrogen bonds with the protonated phenol-O and carbonyl-O atoms °. Complex 5 crystallizes as a trimer in the trigonal system, space group P−3. Each ligand acts as a dibasic pentadentate ligand being shared by two different pentacoordinated Cu(II) metal centres ; ]. Both carbonyl oxygen atoms are deprotonated since enolization and deprotonation of the amide group occur upon coordination, decreasing the C15-N14 bond by 0.02 Å and increasing the C15-O17 bond by 0.026 Å . χm , after diamagnetic contribution subtraction, as well as the product χmT vs. temperature. The χmT product is practically constant over all the temperature range, varying between 0.86 and 0.97 cm3 K mol−1. Assuming the spin-only model with S = ½ (g = 2) for each of three non-interacting Cu(II) ions, the value expected for χmT is 1.25 cm3 K mol−1 for all spins aligned with the external applied magnetic field (↑↑↑) and 0.38 cm3 K mol−1 for a configuration of Cu(II) spins with total spin S = ½ (↑↓↑). The experimental result is far from these two values, indicating the need to consider interactions between the copper spins. Different approaches were considered to fit simultaneously χm, χm−1, χmT vs. T curves, and program PHI  ions, either pointing to a weak isotropic parallel coupling (0<J<1) cm−1 (J < 0) cm−1. The small value obtained for J can be explained by the indirect bonding between the Cu(II) ions.The results indicate a rather weak interaction between the three <1) cm−1 , or to aS = ½ Cu(II) ions at the same distance from each other, in equivalent local symmetry sites, in perfect agreement with the Cu equilateral triangle configuration obtained from crystallographic data.Both spin configurations are consistent with three 1, 5, 6 and 7) electron paramagnetic resonance (EPR) spectra were recorded at 120 K on amorphous  powder samples, and these are reported in g values of 2.114, 2.118, 2.115 and 2.110, respectively. The spectrum recorded on the crystals of [Cu(L5)]3 (5) (with g = 2.112) is very similar to that collected on the polycrystalline powder of the same compound and this demonstrates that the two structures are comparable. The detected isotropic absorptions are usually the result of the dipolar interaction and intercentre exchange between neighbouring units that broaden the hyperfine lines (For selected complexes (ne lines  and suppne lines .IIa and coincide with those of the species that the spectrophotometric titrations interpreted as [Cu(L)] (see infra). Considering that in the X-ray structure of complex 5, the L5 ligand adopts a trinuclear coordination with the donor set , the resonances IIa could be assigned to a complex with equatorial coordination of . In other words, in DMSO and H2O the equilibrium (DMSO)] coexist (see 2O)] is favoured ]xist see , while i3 complexes, the bridges are strong enough to survive to some extent in DMSO solution and are not broken; this is probably due to the nature of the bridge, disclosed by the SCXRD study, which shows a tridentate behaviour of the ligand L on one side, with the set , and bidentate on the other side with the donor set . In aqueous solution we expect the predominance of the [Cu(L)(H2O)] species, as shown in the next section.It is worth noting that, in contrast with other reported polynuclear Cu(II) complexes, which in organic solvents give the mononuclear units , in our Ka values one can determine the actual chemical form and charge at a given pH. The pKa values for the related 8-hydroxyquinoline-hydrazone conjugates (L1-6) were already determined in our previous work  have negligible influence on these pKa values. For L6 with the additional OH group also three pKa values were obtained, although pKa2 and pKa3 were attributed to the two hydroxyl moieties, and the pKa of the benzohydrazide NH was fairly high (>12)  .7 and L8 have one more dissociable proton than L1, namely, L7 has the ammonium group and L8 the pyridinium nitrogen, which may dissociate in the acidic pH range  and the pyridinium nitrogen (L8) are assumed to take place in the same pH range as the quinolinium NH+, it is not possible to assign the pKa1 to a particular moiety, while pKa2 and pKa3 undoubtedly belong to the hydroxyl and the benzohydrazide NH groups, respectively. Despite the relatively small differences found, L7 and L8 are also present in solution in their neutral H2L form at physiological pH, similarly to ligands L1-L5  of both ligand precursors was also determined at pH 7.4 in water at 0.1 M KCl ionic strength  ions were studied by UV-Vis spectrophotometry in 30% (v/v) DMSO/H2O using relatively low concentrations (cL = 50–60 μM), due to the limited water solubility of the ligand precursors as well as of the Cu(II) complexes  system (1 and Cu(II) ‒ L6 systems in 5 in Ka values of the complexes are collected in +, [Cu(L)], and [Cu(LH‒1)]‒ (and [Cu(LH‒2)]2‒ in the case of L6) compositions, and their computed molar absorbance spectra are shown in vide infra).The complex formation processes of L) system . The sol+ species most probably the ligand coordinates via the  donor set of the 8HQ scaffold, while the benzohydrazide NH moiety is still protonated. Considering the much lower pKa value of [Cu(LH)]+ , upon the deprotonation of [Cu(LH)]+ the binding of the benzohydrazide N2im donor atom is highly probable in the species [Cu(L)], adding to  donor set, also in agreement with the SCXRD structure that showed one of the ligands assuming this binding mode. It is worth noting that an equilibrium between [Cu(L)] and the trinuclear complex [Cu(L)]3  is possible; however, under the conditions used for the UV-Vis titrations , the percent amount of polynuclear species is assumed to be rather low. [Cu(LH‒1)]‒ is most likely a mixed hydroxido complex (=[Cu(L)(OH)]‒ with  binding mode) in the case of L1 and L5, while it is formed by the deprotonation of the additional OH group of L6 based on the much lower pKa of [Cu(L)]. Additionally, the development of a new band at 368 nm upon the formation of [Cu(LH‒1)]‒ from [Cu(L)] also supports this suggestion (1 and L5. Formation of [Cu(LH‒2)]2‒ was only found with L6 and this species is suggested to be a mixed hydroxido species.In the [Cu(LH)]Cu(LH)]+  than thaggestion , which i1 and L5, whereas L6 forms somewhat lower stability complexes in comparison to the other two ligands, which suggests that the benzohydrazide N2im donor is not involved in the coordination in complex 6. These Cu(II) complexes are the most stable species at physiological pH, with a level of dissociation at 1 μM concentration corresponding to only ca. 5% for complex 6.Concentration distribution curves  show tha4 and L6 was also studied with oxidovanadium(IV) the speciation data obtained here for the Cu(II) complexes were compared and detailed in the SI (As the complex formation of ligands L2O in the systems containing L4 or L6 with molar ratio 1:1.The Cu(II) binding modes in solution were studied by EPR spectroscopy at 120 K in a mixture of 80% (v/v) DMSO/H6 are depicted in + with the coordination of 8-hydroxyquinoline moiety and  equatorial donor set. Its first three parallel resonances are indicated with I in The spectra measured with LIIa in gz = 2.273 and |Az| = 169.5 × 10−4 cm–1. For this species, whose composition is [Cu(L)] according to the spectrophotometric data, several coordination modes could be possible:   or . The values of ΔGaqcalcd are −9.0 kcal mol–1, –2.3 kcal mol–1, and –3.4 kcal mol–1 ; H2O] is the most stable since it shows the lowest value of ΔGaqcalcd. This is in agreement with the SCXRD structure that suggests that each Cu(II) ion is bound to the same donors from one molecule, and with the EPR spectra collected after dissolving the solid compounds in DMSO solution  is the only one that makes possible the bidentate coordination  to an adjacent [Cu(L)] unit. It must be noted that [Cu(L)] predominates in solution from pH 6 to pH 8, in line with the distribution curves in 3 appears (2O mixtures as well (but is shifted towards the right), and that the species [Cu(L)]  and [Cu(L)]3 coexist in solution. As equilibrium in 3 by spectrophotometry is difficult (see Section 3.6).Upon increasing the pH, another complex is observed IIa in  with gzal mol–1 , respecttion see . Moreove appears , indicat−, N, N2im); H2O] allows also accounting for the appearance of another species at pH higher than 8.0 (III in –1)]–. It has gz = 2.223 and |Az| = 182.0 × 10−4 cm–1. It is plausible that this latter species is originated from [Cu(L)] upon the deprotonation of an equatorial water molecule to give the binding mode ; the coordination of an OH− in the equatorial plane of Cu(II) ion, which replaces a water ligand, agrees well with the decrease of gz and the increase of Az.The formation of the complex [Cu(L)] with coordination [4]2–, denoted with OH in Finally, the deprotonation with a p4 is shown in 2+ ion, are those indicated by I. The EPR parameters are comparable to those of the similar species detected with L6 and the same equatorial coordination mode, , is assigned ; H2O], is assigned. The species IIb has gz = 2.217 and |Az| = 183.6 × 10−4 cm–1. For this species, the superhyperfine coupling with 14N is detected; the number of lines (five) with a ratio 1:2:3:2:1 and a mean coupling constant Az (14N) of 14.8 × 10−4 cm–1 indicate the binding of two nitrogen atoms to copper ] or in a tridentate mode with the binding set  without the coordination of the negative CO− donor group. Moreover, it must be observed that, in the pH range 5.5–8.5, the presence of a minor amount of [Cu(L)]3 in equilibrium with [Cu(L)] cannot be excluded ]–, found by spectrophotometric titrations. Its coordination set is proposed as , similarly to what was detected with L6. IIIb coexists with IIIa, and its detection could be explained by the transformation of [Cu(L)] with  donor set to [Cu(LH–1)]– with . At pH > 12, the resonances of [Cu(OH)4]2– (OH) are observed.Above pH 10, two new sets of resonances are revealed, indicated by and IIIb . IIIa ha−, N, N2im).Overall, both spectroscopic techniques agree concerning the pH speciation of the system. EPR also evidences the coexistence of mononuclear and trinuclear species, which spectrophotometric techniques are unable to disclose due to the similar stoichiometry of both. At physiological pH the complexes are in the neutral form [Cu(L)] and the coordination probably involves the tridentate ligand with the donor atoms  were investigated by cyclic voltammetry in 9:1 (v/v) DMSO:HEPES  with 0.1 M TBAN background electrolyte. These conditions were chosen to provide sufficient solubility of the complexes (1 mM), even though the pH scaling differs from that used for the UV-Vis titrations. Normalized cyclic voltammograms are shown in Copper is a redox-active metal that can react with intracellular reducing agents and induce the formation of ROS. These processes may change the redox state of the cell and impact several important metabolic and signalling processes, which in the end may lead to cell death. The redox properties of selected compounds (kobs) were calculated as the slope of the ln (A/A0) vs. time plots (see an example in 5 with GSH) and the calculated values are collected in 1 ∼ 6 > 5.The direct redox reaction between the copper complexes and GSH and AA was monitored spectrophotometrically under anaerobic conditions at pH 7.4 in the presence of 5% (v/v) DMSO using a tandem cuvette. After the addition of both reducing agents  to the Cu(II) complexes significant spectral changes were observed, and the redox reactions reached the equilibrium within 20 or 60 min for GSH and AA, respectively . These iUV-Vis absorption spectra of the compounds were measured in an aqueous buffer  by dilution of DMSO stock solutions . The comThe analysis of the interaction of newly synthesized compounds with bovine serum albumin (BSA) is the first model in the evaluation of their transport in blood, upon administration. It was monitored by studying the quenching of albumin’s fluorescence emission with increasing concentrations of the complexes . The SteThe ability to interact with DNA was studied for selected compounds with different techniques, which are detailed in SM see : DNA the7  assay . Therefore, the treatment of cells with copper compounds resulted in cell survival loss, albeit with varying efficacy.Copper compounds were tested, along with the free ligands and cisplatin as the positive control, for their cytotoxicity on both melanoma (A-375) and lung adenocarcinoma (A-549) cell lines in the concentration range 0.78–50 μM. Following 72 h of incubation, cell viability was measured B) assay . Comparell lines . Moreove1-L6) were also cytotoxic at µM doses  and 3 was selected to compare the metal effect since the vanadium complex of ligand L3 was the most effective in the melanoma cell line  complexes of the same ligands , but not others . Therefore, this data suggested that while 1, 3 and 8 potentially act through generation of •OH and HOCl, 5 seems to act through other intermediates. Indeed, it is also possible that the cytotoxicity induced by 5 is not by ROS, despite the induction observed in Additionally, to assess whether the ROS induction contributed to the cytotoxicity triggered by these complexes, we used a ROS scavenger, N-acetyl-cysteine (NAC), in combination with  culture , in both1, 5 and 8 exhibited the highest levels of DSB induction. Interestingly, while 3 was able to induce a significant amount of ROS (60% positivity), the amount of DNA breaks did not correlate with the ROS levels, suggesting that the oxidative species generated by 3 may be more efficiently cleared in cells compared to those generated by other complexes. For complex 1 the opposite effect was observed and despite its lower ability to induce ROS formation, the DSB were the highest among the group.Indeed, we examined whether DNA nicks induced by the complexes on pure plasmid DNA would reflect in cell culture as increased DNA break formation. To test this, A-375 cells were stained using γH2AX, which is a common marker for DSBs . Mostly 8 appeared as the most effective drug among the complexes tested herein  as well as 6 and 7 and their ligand precursors were further investigated to reveal their antibacterial activity on the Gram-negative Escherichia coli and Klebsiella pneumoniae strains and the Gram-positive S. aureus using the sensitive (ATCC 25928) and the methicillin-resistant (MRSA 272123) subspecies  and 50 μM (MRSA) MIC values. It should be noted that Staphylococcus aureus causes a wide variety of clinical diseases, and the resistant MRSA subspecies is responsible for several community and hospital-acquired infections (The Cu(II) complexes selected for cell studies  were synthesized as well as eight new Cu(II) complexes by reaction of the ligand precursors (Ln) with Cu(II) acetate. All compounds were characterized and the crystal and molecular structure of both L5 and its Cu-complex were obtained by SCXRD. The trinuclear structure was established for [Cu(L5)]3, and a similar formulation is proposed for [Cu(Ln)]3  in the solid phase. For 1 and 4 the presence of acetate in the solid-state structural formula of the complexes, precluded the formation of the oligomer.Several new benzoylhydrazones  DMSO/H2O. L1-L8 are present in solution in their neutral H2L form, and species [Cu(LH)]+, [Cu(L)] and [Cu(LH−1)]− form in the Cu(II) − ligand systems , and also [Cu(LH−2)]2− in the case of L6. Their formation constants were determined, and their binding modes were established by analysis of the spectrophotometric and EPR data, as well as by DFT methodologies. [Cu(L)] predominates at physiological pH and the relative amount of trinuclear species is not expected to be relevant in the low µM range of concentrations.All compounds present low solubility in water, but they are moderately soluble in DMSO, thus most studies in solution were carried out in media also containing DMSO. Solubility, lipophilicity and proton dissociation constants were determined for all ligand precursors, and for the copper(II) complexes formed by LThe direct redox reactions between these copper complexes and the physiological reducing agents GSH and AA were also evaluated; it was found that the reactions producing Cu(I) species proceed faster with GSH than with AA. Our data also suggests the release of the free ligand during the redox reaction, and that probably the generated Cu(I) complex also dissociates; Cu(I) complexes probably form with GSH (or AA), which are present in a high excess when compared to the 8HQ-containing ligand.1, L5 and L6 pCu values (these reflect the binding strength of the ligands) were computed at 1 μM total metal ion, and two different ligand concentrations at pH 7.4. The pCu values are in the range 7.3–9.0 (for cL = 1 μM) and 9.4 – 12.7 (for cL = 10 μM). These Cu(II) complexes may be considered as high stability species and the concentration distribution curves show that [Cu(L)] species predominate at physiological pH.Based on the stability constants, for the systems with L50 values are in the low µM range. Moreover, our data show that whilst the Cu complexes under study can induce ROS and DSBs in both melanoma and lung adenocarcinoma cells, their ability to induce an apoptotic form of cell death varies, potentially as a result of different clearance mechanisms or efficiencies within the cells. In terms of induction of apoptosis, 3 was the second most effective complex tested in our analyses, despite its lower efficiency in triggering DSBs. This was consistent with our previous report since the oxidovanadium(IV) complex of L3 was the most effective among L1-L6 complexes. Considering the IC50 values, the ability to induce ROS formation, double-strand breaks and cell-death by apoptosis, within the set of compounds tested, 8 seems to be the most promising complex.All complexes appeared to be potent on cancer cells, albeit with varying efficiencies in cell lines of different origins. Globally this group of complexes is more active against the melanoma cells (A-375), than in lung (A-549) cancer cells, but in both cases the IC50 values and macromolecules’ binding parameters), but in general no correlations were found. For the complexes there seems to be some correlation between higher aqueous solubility and higher biological activity, but we do not have sufficient data to clearly state this. The forces driving the interactions and biological effects cannot be easily explained by the electronic effects of the different substituents in the ligands. The only effect clearly seen is that the presence of nitrogen atoms impacts positively the biological activity more than the presence of oxygen atoms and, concerning the presence of halogens, that the F-containing compounds are more active than those with Cl. Moreover, groups with H-donor ability seem to perform better, especially in the lung A-549 cancer cells.Having developed ligands with different substituents we tried to establish a SAR analysis and correlate chemical properties  with the compounds’ biological activity  and MRSA (50 μM), with complexes 1 (50 μM) and 3 and 5 (100 μM) also showing an effect in the same bacteria.Interestingly, complex


The resultant products were characterized using FT-IR, SEM, EDS, and XRD spectroscopy. Electrical conductivity was measured using a four-probe technique, while band gap was calculated via Tauc plots. The results exhibited a significant rise in electrical conductivity of insulating virgin PFR from 2.6 × 10−4 Scm−1 to 8.2 × 10−1 Scm−1 with a 40 and 20 w/w% blend of Ag and graphite in PFR. This synergism was exhibited because graphite and Ag NPs supply excellent junctions for building networks. Both tend to coalesce due to van der Waals forces and high surface energies. Therefore, conductive pathway numbers can be increased, and the contact area can be effectively enlarged. This ternary composite exhibited the lowest bandgap energy value, i.e., 3.1 eV. Thermogravimetric temperature values T0 and Tdeg were increased up to 120 °C and 484 °C, respectively, showing a significant increase in thermal stability. Therefore, the resultant nanocomposite material has good potential to be employed as an ECA in the electronic industry.This study describes the preparation and evaluation of phenol–furfural resin (PFR) from bagasse and its nanocomposites for electrically conductive adhesive (ECA) application. PFR was prepared with furfural extracted from bagasse using a modified acid digestion method. Three different formulations of PFR nanocomposites with conductive nanoparticles, i.e., PFR-silver, PFR-graphite, and PFR-silver + graphite, were prepared using 20, 40, and 60  The current world economy is affected by the scarcity of basic human necessities like clothing, shelter, domestic fuel, and food due to feedstock reserve depletion. Experts have been investigating different options for exploring alternate means for achieving better key solutions for such basic needs. To reduce the world’s dependency on fossil fuels, the implementation of alternative sources such as fuels from renewable resources, production of green materials, and chemicals is now gaining strength ,2,3,4.−1 through using about 80% of graphite along with epoxy resin, resulting in improved mechanical and electrical performances of epoxy–graphite composites [1 and 1 × 106 Ωcm for SWCNTs and MWCNTs. In another work, Dhakate et al. [−1. Nayak and colleagues [1 Scm−1 as the electrical conductivity for epoxy/single-walled nanotubes of carbon-based ECAs. Tang et al. [−6 Ωm and was then applied as the conductive silver ink. Qian et al. presented ECAs with higher conductivity and a lower content of conducting filler through utilizing micro- and nano-scale fillers’ synergistic effects. They investigated this using silver microns coated on copper microns  as ECAs. In the binary ECA compound, the bulk resistivity achieved was 9.42 × 10−5 Ωcm with a 1:9 mass ratio and filler content of only about 60 wt% [3C2Tx nanosheets through simple blending with epoxy resin. Different properties like electrical conductivity, mechanical strength, and both tensile and impact strength were about 4.52 × 10−4 S/cm, 66.2 MPa, and 24.2 kJ/m2, respectively [−6 Ωcm and 6.121 MPa of mechanical strength, with about 170 °C curing temperature in 100 min with 23.4 vol% of filler content [−3 Ωcm, which has the potential to be used as conductive ink [With the advancement of science and technology, there is a need for the development of electronic units that are portable and very small in addition to having highly integrated functions . Consequmposites . The resmposites  studied e et al.  used gralleagues  extensivg et al.  designedt 60 wt% . Ailing ectively . The mec content . GiorgioIn the present work, the authors report the preparation of ECAs through the incorporation of nano-conductive fillers (graphite and Ag) into a matrix of PFR. The resultant nanocomposites were investigated using several characterization techniques including SEM/EDS, FTIR, and XRD, while thermal and electrical properties were investigated as well. The main aim of this research is to synthesize bio-based composites involving feasible and cost-effective methods for its synthesis. Therefore, such conductive resin made from renewable biomass has the potential to be used as a conductive adhesive (ECA), which can be then utilized on an industrial level in parts of electronic equipment as well as electrically conductive inks. To the best of our understanding, these Ag/graphite composites embedded in PFR have not been explored yet .3·6H2O (99%), HCl (37%), NaCl (99%), KOH (99%), Phenol (99%), Urea (99%), Hydrazinium chloride (99%), Silver Nitrate (99%), and Graphite (dry battery cells) were used. All chemicals were procured from Daejung, Korea, while deionized water was used for all solution preparations.Sugarcane plant was purchased from the local market of the city of Wah Cantt, Pakistan. It was first washed and then allowed to pass through a sugarcane presser to obtain bagasse. Subsequently, this bagasse was shade-dried for about 14 days followed by grinding and drying in an oven at about 50 °C for half an hour to ensure it was moisture free. Chloroform (99%), AlCl3.6H2O was prepared. Next, 12% HCl solution along with 2 w/v% of the catalyst material was used as an extraction medium. Bagasse was charged in 1:10 proportion of extraction medium; it was then mixed and agitated to ensure proper mixing of solution and powder. Subsequently, the prepared material was poured into a 1000 mL round-bottom flask attached with a condenser and heated at 100 °C for 2.5 h with magnetic stirring in an electrothermal heating mantle. After bagasse digestion, the distillate was transferred into a separating flask. Chloroform was added into the distillate and shaken well to extract furfural into the chloroform phase. Evaporation of chloroform, via rotary evaporator yielded 53% furfural.A mixture of catalysts comprising equal quantities of NaCl and AlClPhenol furfural resin was prepared according to the method reported by Zeeshan et al. . FurfuraThe graphite rod of an AAA battery cell, having a weight of about 3 g and diameter and height of 0.41 × 1.75 mm was dismantled and ground, followed by sieving using a 0.3–0.4 nm pore-sized sieve. This fine powder was used for the preparation of graphite conductive composite synthesis.3 was dissolved in 40 mL of DI water and kept at 90 °C for 5 min with vigorous stirring. Aqueous solutions of sodium hydroxide (0.64 g/10 mL DI water) and hydrazinium chloride (0.45 g/10 mL DI water) were prepared and simultaneously added into silver nitrate solution, where strong effervescence was noticed. This reaction was allowed to continue for about 1 h until the fizziness disappeared. The obtained precipitates were washed with DI water 5 times at a speed of 5000 rpm to remove any kind of unreacted material and were dried in a vacuum oven at 60 °C for 24 h to obtain greyish silver nanoparticles.A simple approach of chemical reduction method  was usedThe graphite rod of a 3A battery cell was dismantled and ground, followed by sieving using a nanometre-pore-sized sieve. This fine powder was used for the conductive nanocomposite synthesis.w/w ratios of graphite and PFR resin with the sample codes G-20, G-40, and G-60, respectively. For the impregnation of graphite particles, a mechanical stirring process was performed at 25 °C for 25 min. Afterward, samples were placed in a vacuum oven at 90 °C for 24 h for complete drying. When the concentration of graphite powder was increased above 60 w/w ratio, saturation of particles was observed, and mechanical stirring became difficult. This results in nonhomogeneous composite formation along with weak interaction between particles and polymers.Graphite–resin composites were prepared in 20:60, 40:40, and 60:20 w/w ratios of Ag and PFR resin.The silver and resin nanocomposites were prepared with the same procedure used for the graphite resin composite preparation. Samples were labelled as S-20, S-40, and S-60 for the compositions as 20:60, 40:40, and 60:20 w/w ratios of silver, graphite, and PFR resin, respectively.For ternary composite preparation, both graphite and Ag nanoparticles were used as per the aforementioned method. These are coded as SG-20, SG-30, and SG-40 for 20:40:40, 30:30:40, and 40:30:40 −1 to study the characteristic peaks of furfural and resin. A KBr disc was used as sample holder material. A BRUKER D8 advanced X-ray diffractometer  equipped with Cu K alpha 0.154 nm wavelength radiation sources was employed in theta range of 10 to 80° to examine the crystallinity of the filler materials. The surface topography of the nanocomposites was examined using a JSM-6490A, JEOL  SEM instrument, while images were captured at resolution scale of 10 µm and 1 µm. Thermogravimetric analysis (TGA) was conducted via an SDT 650 TA instrument  under inert conditions. Initial temperature was 26 °C and was increased to 900 °C with a 10 °C/min ramp rate in an inert nitrogen atmosphere. Jasco V-770 via UV-Visible Spectrophotometer  was used for the bandgap measurements via the diffuse reflectance spectroscopic technique (DRS). Conductivity of the samples was measured using a four-point probe method via JANDEL RM3000  test unit. The technique was used to measure the resistivity of the samples from which the conductivity was calculated through taking the reciprocal of resistivity. A DC supply with constant current was used for all the measurements.A Perkin Elmer Spectrum 100 FT-IR Spectrometer  was used in the frequency range of 350–4000 cm−1 in the FTIR spectrum, which is due to the Ar-H stretch, as shown in −1 and 2807 cm−1 [−1 is due to a C=O stretch. Moreover, the two bands at 1566 cm−1 and 1462 cm−1 are corroborated by the presence of an aromatic ring C=C bond. The presence of a furan ring was ensured by a 1215 cm−1 bond stretch, while the 1014 cm−1 band stretch reflects the presence of a C-H plane. A=C-H out-of-plane bending vibration and C-H out-of-plane (monosubstituted) bending vibration are exhibited at 929 cm−1 and 740 cm−1, respectively [−1 due to -OH  = 2.4 Å, 44.0° d(200) = 2.1 Å, 64.0° d(220) = 1.5 Å, and 77.1° d(311) = 1.2 Å. Meanwhile, 2θ values were found at 38.1°, 44.0°, 65.1°, 77.1° and 38.1°, 44.1°, 64.2°, 77.1° for S-40 and S-60, respectively, having similar diffraction planes and d-spacing values to of pure Ag nanoparticles. These results declare that even after the mixing of silver nanoparticles in the resin, no silver oxide such as Ag2O is produced, thus confirming the successful blending and formation of this Ag-PFR nanocomposite.Pure silver and its nanocomposites were characterized via XRD . Ag nano(002) = 3.4 Å, 38.0° d(111) = 2.4 Å, 44.2° d(200) = 2.0 Å, 54.5° d(004) = 1.7 Å, 64.3° d(220) = 1.4 Å, and 77.3° d(311) = 1.2 Å, while the SG-40 composite has almost similar results of 26.4° d(002) = 3.4 Å, 38.0° d(111) = 2.4 Å, 44.2° d(200) = 2.0 Å, 54.5° d(004) = 1.7 Å, 64.2° d(220) = 1.5 Å, and 77.3° d(311) = 1.2 Å. The composite SG-60 has shown diffraction signals at 26.4° d(002) = 3.4 Å, 38.0° d(111) = 2.4 Å, 44.2° d(200) = 2.0 Å, 54.4° d(004) = 1.7 Å, 64.3° d(220) = 1.4 Å, and 77.3° d(311) = 1.2 Å. These results  was 96 °C, while decomposition temperature Tf (where decomposition ended) was 727 °C. Tdeg was 438 °C, the temperature at which maximum degradation was achieved. Likewise, the TGA values of pure graphite were 600 °C and 705 °C for T0 and Tf, respectively. Its thermal stability range was 26 °C to 600 °C. Degradation temperature increased from 438 °C to about 500 °C as the graphite powder was added. Detailed TGA results of binary composites of resin and graphite are described in Thermal stability behaviour was checked for all pure resin, nanoparticles, and nanocomposites via TGA. For pure resin, onset temperature Tf, T0, and Tdeg were 883 °C, 200 °C, and 549 °C, respectively, for silver nanoparticles. After blending with resin, their thermal stability was rechecked. It was observed that T0 values increased from 96 °C to 117 °C, 127 °C, and 122 °C for S-20, S-40, and S-60, respectively, because of silver nanoparticles which have increased thermal stability. Similarly, Tdeg values also increased from 438 °C to 499 °C, from resin to S-60, confirming thermal stability increases due to the incorporation of nanoparticles (articles .0 values of resin increased from 96 °C to up to 120 °C for all ternary resin composites., whereas Tdeg values increased from 438 °C to 474 °C, 484 °C, and 512 °C for S-20, S-40, and S-60, respectively (ectively . These rA diffuse reflectance spectroscopic technique was used for the measurement of electrical conductivity. Band gap calculations were performed using Tauc plot calculations. Pure graphite extracted from dry battery cells has exhibited a bandgap energy value of about 3.5 eV. A similar value was reported by other researchers in the literature , while rPure silver nanoparticles have exhibited a bandgap energy value of about 3.5 eV. A similar result was reported by AJ Das et al. . All thew/w% of graphite loading that has synergistically performed for electronic conduction, while 40 w/w% of resin aided perfectly in providing adhesive, fine, and uncracked cured textured samples. SG-40 has exhibited the lowest bandgap because of good conductive path formation due to the synergistic effects of graphite and Ag filler particles when incorporated with PFR. These nanocomposites with a lower bandgap, better thermal stability, and increased electrical conductivity have the desired properties for use in parts of electronic applications and can be applied as conductive ink due to the lower bandgap energy value. Therefore, this lowered bandgap energy value and best electrical conductivity have the potential to be applicable at an industrial level.The lowest bandgap energy value, i.e., 3.1 eV, was exhibited by the SG-40 c Ag-grap−4 Scm−1, but conductivity significantly increased up to 2.2 × 10−2 Scm−1 by 20 w/w% of graphite–resin composite. This value of electrical conductivity was further increased to about 5.6 × 10−2 Scm−1 and 3.92 × 10−1 Scm−1 with 40 w/w% and 60 w/w% of graphite resin composite, respectively. Upon further addition of graphite, mechanical stirring is hindered, resulting in self-agglomeration and lump formation due to voids and spaces in the graphite powder.The electrical conductivity of the composites was indirectly measured using a four-probe method. Two probes at adjacent corners transfer a constant supply of current, while the remaining probes measure the difference in voltage between the two corners. −1 Scm−1, 2.75 × 10−1 Scm−1, and 3.92 × 10−1 Scm−1 upon addition of 20 w/w%, 40 w/w%, and 60 w/w% of Ag nanoparticles, respectively, which is far greater than the conductivity results of Ag NPs in resin of 2.48 × 10−2 Sm−1 reported by Ibrahim et al. [−1 Scm−1 as it consists of both silver and graphite nanoparticles in optimum concentration, while 4.12 × 10−1 Scm−1 and 5.98 × 10−1 Scm−1 were calculated for SG-20 and SG-30, respectively. The optimized composite SG-40 comprising 40 w/w% Ag and 20 w/w% of graphite furnished the best conductivity results.To study the electrical properties of resin, silver nanoparticle metallic filler was also loaded in different percentages. The conductivity of Ag-resin nanocomposite film was calculated as 1.72 × 10m et al. . These rThis four-point probe technique also confirms that there exists a direct relationship between the added content of conducting nanoparticles up to a certain point, along with the decrease in bandgap energy values, and increased thermal conductivity in all the resin composites. Such nanocomposites with increased electrical conductivity, better thermal stability, and decreased bandgap energy value have the ability to be applied in numerous electronic parts and as conductive ink. Therefore, such composites have the potential to be applicable at the industrial level. However, further enrichment causes nonhomogeneous mixing as well as difficulty in mechanical stirring. It is proposed that both silver and graphite nanoparticles act as a junction in between polymeric chains of resin.A renewable, native, and economical material, i.e., bagasse, was thrivingly used to extract furfural and then employed in green resin synthesis which served as the adhesive material between conductive fillers and the surface. Three distinct ratios of conductive fillers were loaded in the polymerized matrix to obtain the optimized adhesion and electrical performance of films. Various hybrids of nanocomposites were corroborated by the results of TGA, SEM, XRD, FTIR, four-probe technique, and EDS. Results showed a significant increase in the conductivity of pure resin and that of nanocomposites; an abrupt increase was observed when both nanoparticles were added. TGA, four-probe, and bandgap results have shown that PFR synthesized from bagasse can be loaded with conductive nanoparticles. Therefore, it is proposed that graphite and silver nanoparticles may act as the conducting junctions in between the different polymeric chains of resin, which ultimately increases thermal and electrical conductivity. So, it has good potential to be employed as an electrically conductive adhesive.


Matricarla chamomilla L. is native to European countries and widely cultivated in China, especially in Xinjiang. It has been used in Uygur medicine for the treatment of cough caused by asthma. In this study, UHPLC-Q-Orbitrap-MS was used to detect and identify the components from the active fraction of M. Chamomile, 64 compounds were identified by combining the standards, related literatures and mass spectrometry fragments, including 10 caffeoyl quinic acids, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. Furtherly, the anti-asthma activity of active fraction of M. Chamomile was investigated in OVA-induced allergic asthma rat model. The results showed that the number of EOS in Penh and bronchoalveolar lavage fluid (BALF) in the group of the active fraction of M. Chamomile was significantly lower than that in the model group. Besides, the active fraction of M. Chamomile can significantly reduce the IgE level and increased glutathione peroxidase (GSH-Px) in the serum of OVA-induced rats, and ameliorated OVA-induced lung injury. Hence, M. Chamomile could be used to treat asthma through their in vivo antioxidant and anti-inflammatory effects. This study explored the potential material basis of M. Chamomile for the treatment of asthma. In secondary mass spectra, they all had characteristic fragment ions at m/z 191, 179, and 173. Compounds 1, 2 and 3 were identified as neochlorogenic acid (3-O-caffeoylquinic acid), cryptochlorogenic acid (4-O-caffeoylquinic acid) and chlorogenic acid (5-O-caffeoylquinic acid), respectively, based on the retention times and the agreement of the fragment ions with the standards [Compound 1 (tR = 26.68 min) showed a molecular ion peak at m/z 677 [M − H]-. In the secondary mass spectrum, fragment ions of m/z 515 [M − 162]−indicated that the glucose were lost from molecular ion. Also, di-O-caffeoylquinic acid of characteristic fragment ions m/z 353, 191, 179, and 135 were observed. Therefore, it was presumed that compound 4 was di-O-caffeoylquinic acid-glucoside based on the cleavage characteristics of the compound -. The fragment ion of secondary mass spectra at m/z 353, suggesting that these compounds were decaffeoylquinic acid. Compounds 5, 6, 7, 8 and 9 were identified as 1,3-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid (isochlorogenic acid B), 3,5-di-O-caffeoylquinic acid (isochlorogenic acid A), 4,5-di-O-caffeoylquinic acid (isochlorogenic acid C), and 1,5-di-O-caffeoylquinic acid (isochlorogenic acid C), based on the retention times and the cleavage characteristics of these compounds with the standards. C) [Compound 5 (tards. C) . The cleR = 56.05 min) showed a molecular ion peak at m/z 529 [M − H]-. Characteristic fragment ions of the secondary mass spectra were observed at m/z 367 [feruloylquinic acid-H]-, m/z 353 [caffeoylquinic acid-H]-, and m/z193 [ferulic acid-H]-, m/z 191 [quinic acid-H]-, m/z 179 [caffeic acid-H]-, m/z 173 [quinic acid–H2O–H]-, and m/z 135 [caffeic acid-CO2]-. According to the similarity of the mass spectral cleavage rules and the previous literature -, respectively. The MS2 spectrum showed the fragment ions of compound 41 at were m/z 241, 271, 151, and 133. According to retention time and standard, compounds 41 was identified as luteolin, the cleavage rule was shown in m/z 285 was shown in the secondary mass spectra of compounds 11, 25, 26 and 28, which was the characteristic fragment ion of luteolin. Therefore, Therefore, this confirmed the mother nucleus of these compounds was lignocerebrosides. Based on the retention times of compounds 11 and 26 and the fragment ions with the standards, these two compounds were identified as lignocerebroside-8-C-glucoside and lignocerebroside , 13 (tR = 18.09 min), 15 (tR = 21.47 min), 18 (tR = 22.27 min) and 20 (tR = 22.67 min) had same molecular ion peaks at m/z 463 [M − H]-. The fragment ion at m/z 301 and 300 indicated that these compounds contain glucose group. The fragment ions at m/z 255, 227, and 151 indicate that the parent nucleus was a quercetin glycogen. Therefore, these compounds are presumed to be quercetin-O-glucoside and its isomers. Based on the above mass spectral information and the agreement of retention times with the standards, compounds 12, 13 and 15 were deduced to be quercetin-3-O-glucoside (isoquercitrin), chrysin, and quercetin-7-O-glucoside, respectively - ion at m/z 477, 477, 609, 477, 609 and 301, respectively. 477, 609, 477, 609 and 301 [m/z 301 [M-H-176]-, indicating that all were missing a glucuronide. The fragment ions at m/z 255, 227, and 151 indicated that the parent nucleus is a quercetin. Compounds 16, 19 and 23 are presumed to be quercetin-O-glucuronide and its isomers, respectively, based on the characteristic ions fragment and previous literature -. The fragment ions at m/z 284 [M-H-H-162]- or 285 [M-H-162]- indicated the loss of a glucose. The characteristic ions of kaempferol were m/z 255, 227, 163, and 151. Therefore, these compounds presumed to be kaempferol-O-glucoside and its isomers based on the above mass spectral information and reference -. The fragment ion at m/z 268 as the base peak in the MS2 spectrum, indicating the loss of a glucose. The fragmentation information at m/z 268, 240, 211, 151 were consistent with galangin, hence these two compounds are galangin-O-glucoside and its isomers.Compounds 33 (tR = 39.66 min) had molecular ion 577 [M − H]- and showed fragment ion at m/z 269 as the base peak in the MS2 spectrum, indicating the loss of a rhamnose and glucose. The other fragment ions were m/z 255, 117, 107, and 149, suggesting that the molecular parent was apigenin. Therefore, based on the above information and previous literature reports, the compound was deduced to be apigenin-O-rhamnose-hexacoside.Compound 35 (tR = 47.95 min), 37 (tR = 48.19 min) and 48 (tR = 65.48 min) showed molecular ion peaks at m/z 607, 461 and 299, respectively. According to characteristic fragment ions at m/z 284 [M − CH3]-, 255 [M − CH3 –CO–H]-, 227 [M–CH3–2CO-H]- and 211 [M–CH3– CO-CO2-H]-, compound 48 was identified as methyl kaempferol. Based on the above mass spectral information and references, compounds 36 and 37 had same fragment ion at m/z 299, indicating the loss of desmethyl glucose and glucose, respectively. Therefore, compounds 36 and 37 were identified as methyl kaempferol-O-demethylhexoside and methyl kaempferol-O-hexoside, respectively.Compounds 36 (tR = 51.31 min) and 39 (tR = 51.70 min) have molecular ion [M − H]- at m/z 505 and 491, respectively. In the MS2 spectrum, the characteristic ions of dihydroxyquercetin at m/z 329, 314, 299 and 271. Based on the mass spectral information and previous literature reports, the compounds were tentatively identified as dihydroxyquercetin-glucuronide and dihydroxyquercetin-glucoside.(3)CoumarinsCompounds 38  and 50 (tR = 7.87 min) presented the molecular ion at m/z 341 and 179 [M+H]+, respectively. Compounds 50 had characteristic fragment ions of coumarins at m/z 119 and 91 in the MS2 spectrum. Based on mass spectral fragment information and retention times consistent with the standards, compounds 49 and 50 were deduced to be dihydroxycoumarin-O-glucoside (daphnoside) and dihydroxycoumarin (daphnetin), respectively +, m/z 146 [M + H-CH3OH]+, and the more abundant m/z 133 [M + H-CH3OH-CO]+ can be observed. 133 [M + H-CH3OH-CO]+. Based on the above mass spectrometric information and previous literature, the compound was identified as a hydroxy-methoxy-coumarin and its isomers +, respectively. The fragment ion at m/z 119 and 91 as well as retention time were consistent with the standard, hence compound 53 was identified as coumarins. The MS2 spectra of compounds 54, 55 and 56 all had fragment ions at m/z 145, suggesting that the parent nucleus was coumarin. Based on the mass spectral information and literature reports, compounds 54, 55 and 56 were deduced to be hydroxycoumarin, hydroxycoumarin (inocyanin) and methoxycoumarin, respectively +, and the structural formula was C10H10O2N. The fragment ion at m/z 117 [M + H-59]+ was observed in the MS2 spectrum, indicating the loss of the acetic acid. Therefore, the compound was deduced to be indole-acetic acid.Five indole alkaloids were identified in the active fraction of lkaloids . CompounR = 4.60 min) and 60 (tR = 5.37 min) had molecular ion peaks [M+H]+ at m/z 146, and the structural formula was C9H6ON. The fragment ion at m/z 118 [M + H-28]+ was observed in the MS2 spectra, indicating the loss of a formyl group. The fragment ions at m/z 91 and 77 were the characteristic fragment information of the indole-like parent nucleus. Therefore, compounds 58 and 60 are inferred to be indole-carbaldehyde and its isomers.Compounds 58 (tR = 4.16 min) and 61 (tR = 5.09 min) showed same molecular ion peak at m/z 188 [M+H]+, with a structural formula of C11H10O2N. The fragment ion at m/z 146 [M + H-42]+ was observed in the MS2 spectrum, suggesting that the structure contained an acetyl group; and the fragment ion at m/z 118 [M + H-42-28]+, suggesting further loss of the formyl group. Also, indole-like characteristic fragment ions such as m/z 91, 77 were observed. Based on the above information, compounds 59 and 61 were inferred to be acetylindole formaldehyde and its isomers, respectively.(5)Other compoundsCompounds 59 (tR = 1.05 min) and 64 (tR = 8.36 min) showed same molecular ion peaks at m/z 191 [M − H]-, and molecular formulas were C6H9O7 and C7H13O6, respectively. Based on the retention time and fragmentation information with the standard, compound 62 and 64 were dentified as citric acid and quinic acid [Compounds 62  showed a molecular ion peak [M − H]- at m/z 179, and fragment ions were observed in the secondary mass spectrum at m/z 135, indicating the loss of CO2. Based on retention time and mass spectral information consistent with the standard, it was confirmed as caffeic acid [Compound 63 (teic acid .3.3P < 0.005); administration group was reduced in turn compared with the model group in a dose-dependent manner (P < 0.005).The results are shown in 3.4P < 0.01); compared with the model group, Penh of administration groups was reduced significantly in rats (P < 0.01).The results were shown in 3.5P < 0.005), compared with the normal control group; and, compared with the model group, the middle and high dose groups of the active fraction of M. Chamomile could significantly reduce the IgE level in rats .The results were shown in 3.6P < 0.01); the eosinophil count was reduced significantly in BALF of rats in the middle and high dose groups of the active fraction of M. Chamomile, compared with the model group (P < 0.01).The results were shown in 3.7P < 0.005); and compared with the model group, the serum GSH-Px levels of administration group was increased significantly in a dose-dependent manner (P < 0.005).The results were shown in 3.8The results were shown in 4Asthma is a life-threatening chronic airway disease. There are about 300 million people suffering from asthma in the world, and this number is increasing at an alarming level, reaching an annual asthma mortality rate of more than 250,000. T cells associated with asthma are considered to be the key pathogenic factors of inflammatory response in asthma, which release various Inflammatory cytokine, such as interleukin , tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and specific allergens produced by activated B lymphocytes . Goblet M. Chamomile. A rapid and effective systematic identification method of chemical components of chamomile was established the separation and identification of the components in the active fraction of M. Chamomile. The chromatographic peaks of quasi-molecular ions were obtained, 64 compounds were identified by combining the standards, related literatures and secondary mass spectrometry fragments, including 10 caffeoyl quinic acid compounds, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. This study demonstrated that the active fraction of M. Chamomile contained abundant polyphenol compounds, especially flavonoids, which have been said to account for most of the antioxidant activities and anti-inflammatory effects of plant extracts [M. Chamomile were effective components against asthma, especially flavonoids were the important bioactive components, mainly including luteolin, quercetin, kaempferol and their related compounds. Hence, the identification results provide a basis for clarifying the active fraction of M. Chamomile and the quality control of related new drug of anti-asthma development process.In this study, UHPLC-Q-Orbitrap-HRMS coupling technique was used to the separation and identification of the components in the active fraction of extracts ,44. Prevextracts ; luteoliextracts . Flavonoextracts . Chlorogextracts . Coumariextracts . These pM. Chamomile. The results showed that the number of EOS in Penh and bronchoalveolar lavage fluid (BALF) in the active fraction of M. Chamomile was significantly lower than that in the model group. IgE has been the immunoglobulin traditionally linked to an allergic response in conditions such as asthma; and enzyme-linked immunosorbent assay (ELISA) was used to detect the serum IgE level. The active fraction of M. Chamomile can significantly reduce the serum IgE level of asthmatic rats induced by OVA. In addition, compared with the asthma model group, the active fraction of M. Chamomile significantly increased glutathione peroxidase (GSH-Px) in the serum and ameliorated lung injury significantly. The results suggested that the active fraction of M. Chamomile had inhibitory effects on airway inflammation, hyperresponsiveness and oxidative stress in asthmatic rat.Furtherly, the OVA-induced allergic asthma rat model was established for the purpose of investigating the anti-asthma effect of the active fraction of M. Chamomile by UHPLC-Q-Orbitrap-HRMS technology, including 10 caffeoylquinic acids, 38 flavonoids, 8 coumarins, 5 alkaloids and 3 other compounds. Flavonoids were the important bioactive components of M. Chamomile against asthma, especially luteolin, quercetin, kaempferol and their related compounds. And, the active fraction of M. Chamomile possessed potent anti-inflammatory and anti-oxidative effects to exerts protective effects against OVA-induced asthmatic symptoms. Hence, M. Chamomile could be used in the future treatment of asthma, and future research is required to explore the underlying mechanisms.Overall, combing with retention time, mass data, standards and information reported previously, 64 compounds were identified from the active fraction of Conceptualization, X.X. and H.A.A.; methodology, Q.L. R. A. G.X, and X.K; software, X.X.; validation, R.A.; formal analysis, Q.L. and R. A.; investigation, Q.L. R. A. G.X, X.K and F.Z; resources, X.X. F.Z and H.A.A.; data curation, R.A.; writing—original draft preparation, Q.L. R.A. G.X, and X.K; writing—review and editing, X.X. and H.A.A.; visualization, X.X.; supervision, X.X. and H.A.A.; project administration, H.A.A.; funding acquisition, Q.L All authors have read and agreed to the published version of the manuscript.10.13039/501100015310Xinjiang Uygur Autonomous Region Natural Science Fund (2021D01E32).This work was supported by The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Introduction: Triple-combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) was introduced in August 2020 in Germany for people with CF (pwCF) ≥12 years (yrs.) of age and in June 2021 for pwCF ≥6 yrs of age. In this single-center study, we analyzed longitudinal data on the percent-predicted forced expiratory volume (ppFEV1) and body-mass-index (BMI) for 12 months (mo.) after initiation of ETI by linear mixed models and regression analyses to identify age- and severity-dependent determinants of response to ETI.Methods: We obtained data on 42 children ≥6–11 yrs, 41 adolescents ≥12–17 yrs, and 143 adults by spirometry and anthropometry prior to ETI, and 3 and 12 mo. after ETI initiation. Data were stratified by the age group and further sub-divided into age-specific ppFEV1 impairment. To achieve this, the age strata were divided into three groups, each according to their baseline ppFEV1: lowest 25%, middle 50%, and top 25% of ppFEV1.Results: Adolescents and children with more severe lung disease prior to ETI (within the lowest 25% of age-specific ppFEV1) showed higher improvements in lung function than adults in this severity group . In all age groups, participants with more severe lung disease showed higher BMI gains than those with medium or good lung function (within the middle 50% or top 25% of age-specific ppFEV1). Regression analyses identified age as a predictive factor for FEV1 increase at 3 mo. after ETI initiation, and age and ppFEV1 at ETI initiation as predictive factors for FEV1 increase 12 mo. after ETI initiation.Discussion: We report initial data, which suggest that clinical response toward ETI depends on age and lung disease severity prior to ETI initiation, which argue for early initiation of ETI. The advent of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to an unprecedented improvement in the lives of people with CF (pwCF). Licensing of triple-combination CFTR modulator therapy in the form of elexacaftor (ELX)/tezacaftor (TEZ)/ivacaftor (IVA), henceforth termed ETI, increased the availability of CFTR modulator therapy. In Germany, ETI was introduced in August 2020 for pwCF ages 12 and older (pwCF ≥12), and in June 2021 for ages 6 and older (pwCF ≥6), permitting CFTR modulator therapy for more than 85% of patients in those age strata. Until now, determinants of ETI response have not been identified. Previous studies on other CFTR modulators suggest that age at therapy initiation significantly impacts upon response to CFTR modulator therapy.Younger age at initiation of IVA permits better preservation of lung function and a larger impact on pulmonary exacerbation (PEx) . In adolBy proxy, the findings of Nichols et al., which show a significant correlation between sweat chloride reductions and ppFEV1 improvements after 6 months of therapy , might an = 226 people with CF ≥ 6 years of age. We focused on comparing age strata with the similar severity of lung disease prior to ETI initiation, hypothesizing that earlier ETI initiation confers superior benefits with regard to the key clinical endpoints ppFEV1 and BMI.Our study aimed to address determinants of response to ETI through the analysis of 3-month and 1-year follow-up (FU) data from a single-center cohort of PwCF receive standardized diagnostic procedures and treatment in our center, according to national and international guidelines, including 3-month intervals of outpatient visits. All data used for this study were recorded in an in-house electronic patient record at every visit, according to definitions predefined by the German CF Registry.2) were collected prior to and on months 3, 6, 9, and 12 after ETI initiation. Lung function was referenced according to the Global Lung Function Initiative (GLI)  . The BMIWe performed an exploratory, retrospective, single-center, post-approval cohort study. We extracted demographic data and data on ppFEV1 and BMI of all pwCF from our CF center, who received ETI therapy for at least 3 (for pwCF ≥6–11) or 12 (for pwCF ≥12) uninterrupted months. Data for pwCF ≥12 years were collected between August 2020 and January 2023, and data for pwCF ≥6, between June 2021 and January 2023. For patients ≥6–11 years of age, we only included data from FU 3 months in our analyses as the group size of further FU data was increasingly limited due to the recent approval of ETI in this age group.• pwCF ≥18 years: ≤P25: 27.2 (23.4–30.4), P50: 53.1 (45.4–60.7), and>P75: 82.3 (77.0–92.9) for median (IQR)• • pwCF ≥12–17 years: ≤P25: 56.4 (50.2–61.9), P50: 83.4 (73.9–91.3), and>P75: 98.9 (95.5–110.9) for median (IQR)• • pwCF ≥6–11 years: ≤P25: 70.7 (61.4–80.7) P50: 87.7 (82.9–95.0), and>P75: 102.1 (100.6–105.4) for median (IQR)• Data were stratified according to age  and age-group-specific lung function impairment percentiles. We aimed to compare pwCF with similar degrees of lung disease across age groups. Yet, average ppFEV1 values, as a proxy for lung disease severity, are different across these age groups. We, therefore, chose to stratify pwCF according to the average ppFEV1 within their age group as reference, creating age-specific sub-groups of lung disease severity. For this purpose, we chose the common cut-off values of the lowest and highest 25th percentile for baseline ppFEV1 of the age group in question, thereby creating three groups: “severely affected”: baseline ppFEV1 ≤25th percentile (≤P25), “average”: 26th–74th percentile (P50), and “less affected”: ≥75th percentile (≥P75) for the age groups ≥6–11 yrs, ≥12–17 yrs, and ≥18 yrs, respectively. The age-dependent percentiles for ppFEV1 for our cohort were calculated and are also stated in . Initially, measurements were tested for normal distribution. Differences between groups were analyzed by the Mann–Whitney U test or Kruskal–Wallis test, as appropriate. Frequency differences between nominally distributed groups were calculated by a chi-squared test. Data were corrected for multiple testing by Bonferroni correction.All statistical analyses were performed with Statistical Package for Social Sciences . Descriptive data were calculated as the median and interquartile range (IQR)We used a generalized linear model to assess the effects of gender, age group, and prior CFTR modulator therapy on the outcome ppFEV1 or BMI for all pwCF at or above 12 years of age included in our analyses.We performed regression analyses to address the effects of age, gender, and CFTR modulator therapy prior to ETI initiation, as well as baseline values for ppFEV1, BMI, and the severity of lung disease according to the previously defined percentiles at baseline. PpFEV1 or BMI gains at 3 or 12 months were considered outcomes. We included data from all pwCF at or above 6 years of age for gains at 3 months and from all pwCF above 12 years of age for gains at 12 months.p-value <0.05 was considered statistically significant.A All patients or their legal guardians provided consent to the anonymized scientific use of personal clinical data for research purposes, either as written informed consent to participate at scientific studies of the German Center for Lung Research (DZL) registry  and/or the German CF registry .n = 226 pwCF from our center into our analyses. Data were obtained at a median time of 3.1 months for all n = 226 subjects included in the study and 12.4 months for n = 182 (80.5%) subjects ≥12 years after ETI initiation. Median age at ETI initiation was 22.5 yrs (IQR 13.4–30.5), of which 51.8% were female subjects, and 45.1% already underwent CFTR modulator therapy prior to ETI initiation.We were able to include 2 (IQR 0.2–1.5) and +1.4 kg/m2 (IQR 0.4–2.6) at 12 months compared to prior ETI initiation (The median gain of ppFEV1 at 3 months FU was +9.7 (IQR 5.1–17.8) and +9.6 (IQR 5.5–16.4) at 12 months compared to ppFEV1 at baseline. The median BMI gain at 3 months FU was +0.8 kg/mitiation .n = 41) and participants ≥18 yrs of age (n = 143)  .p = 0.001), and BMI absolute values were still significantly lower in the group ≥12–17 yrs of age (p = 0.001).Participants ≤18 yrs of age included a higher proportion of female subjects and used CFTR modulator therapy significantly less prior to ETI initiation. As expected, these patients had a higher ppFEV1 but lower absolute BMI values than participants ≥18 years of age. PpFEV1 at 3 and 12 months of ETI treatment were also significantly higher , while neither gender, prior CFTR modulator therapy, nor BMI or degree of lung disease severity at baseline was associated with this outcome. BMI gains at 3 months were only influenced by ppFEV1 at baseline (p = 0.008), but none of the other parameters were tested.Regression analyses for ppFEV1 gains revealed that after 3 months of ETI therapy, age and ppFEV1 at ETI initiation were significantly associated with ppFEV1 gains . For BMI gains at 12 months, age at ETI initiation did have a significant effect (p = 0.004) as did BMI at initiation (p = 0.039), but contrary to the 3-month data, ppFEV1 at initiation did not associate with BMI gains.Our analyses with only those patients aged 12 yrs and older showed similar trends with regard to the impact of age and ppFEV1 at ETI initiation upon ppFEV1 gains at 12 months after ETI initiation  provided more insights into the critical role of age at initiation of ETI. Younger age is associated with significantly higher ppFEV1 gains at 3 and 12 months after ETI initiation . Neither gender, prior CFTR modulator therapy, nor BMI or degree of lung disease severity at baseline was associated with ppFEV1 gains at 3 or 12 months.Regression analyses of only those patients in the group with the most severe lung disease  and 9.0 (4.6–14.2) vs. 10.5 (5.6–22.0) vs. 9.2 (5.9–14.2) for 12-month FU (p = 0.258) for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively.Given our results on age as a significant determinant of ppFEV1 and BMI gains, we hypothesized that pre-existing lung disease might exert different influences on ppFEV1 or BMI gains, depending on the age group analyzed. We, thus, stratified our cohort for age-specific severity of pre-existing lung disease by calculating age group-specific lung function percentiles (age-specific ppFEV1 ≤25th percentile (≤P25), 26th–74th percentile (P50), and ≥75th percentile (≥P75). This stratification did not lead to statistically significant differences with regard to age, proportion of female subjects, or prior CFTR modulator use . PpFEV1 p = 0.041, for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively]. BMI changes 3 and 12 months after initiation of ETI did show significant group differences , suggesting higher BMI gains in the participants with lower lung function (+1.2 (0.6–2.1) vs.+ 0.7 (0.0–1.4) vs. + 0.7 (0.0–1.3) for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively, at 3-month FU and +2.2 (0.6–3.5) vs. +1.1 (0.3–2.6) vs. +1.1 (0.4–2.3) for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively, at 12-month FU  vs. 19.3 (17.0–21.2) vs. 20.2 (18.0–22.2), p = 0.027 for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively). This difference abated at 12 months post-ETI initiation (+13.8 (11.3–24.3) vs. +10.5 (5.6–24.0) vs. +8.9 (2.9–11.9), p = 0.09 for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively) , which abated at 12 months (7.2 (3.5–11.8) vs. 10.4 (5.6–22.0) vs. 9.3 (6.2–14.2), p = 0.121 for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively)  vs. +12.3 (6.2–21.6) vs. +11.3 (6.0–16.6), p = 0.002 and p = 0.012 for 3 and 12 months, respectively, When comparing adolescents (≥12–17 years) and adults with worse lung function (≤P25) for ppFEV1 gain after 3 and 12 months of ETI initiation, adolescents showed significantly higher improvements at both time points than adults  than adults with ppFEV1 ≤P25, this finding attained significance with a higher gain of ppFEV1 in children (children and adolescents: +19.3 (8.0–27.3) vs. adults: +7.5 (3.7–10.7), p = 0.007) 3 months after ETI initiation (data not shown).Complementing our cohort with data from patients between 6 and 11 years of age supported the finding that in younger patients, those with more severe lung disease show higher gains in ppFEV1 3 months after ETI initiation . At basetively], . Furtherp = 0.034) and 12 months , although these differences were statistically significant only at 3 months post-ETI initiation  (p = 0.089 and p = 0.438), suggesting more significant improvements in response to ETI in those participants with a worse lung function. Indeed, for adults, significantly larger improvements in BMI were observed in those pwCF with the worst lung function after both 3  and 12 months   vs. 20.5 (18.6–22.3) vs. 21.7 (20.2–24.3), ctively) . These d= 0.048) .p = 0.026 for ppFEV1 ≤P25 vs. P50 vs. ≥P75, respectively).Interestingly, in pwCF ≥6–11 years of age , BMI impOur analyses provide initial data, which suggest that ETI response, as reflected in ppFEV1 and BMI levels, is dependent on age at initiation and lung disease severity. Specifically, the data show that children and adolescents with more severe lung disease (defined as age-group-specific ≤25% percentile ppFEV1) exhibited higher ppFEV1 gains at 3 and 12 months after ETI initiation than those with a better lung function at baseline in the same age group. Furthermore, inter-age comparisons of those participants affected most severely with respect to lung disease (age-specific ppFEV1 ≤P25) showed higher ppFEV1 gains in children and adolescents than adults at 3 and 12 months after ETI introduction. These findings are supported by regression analyses in the sub-group of all pwCF affected most severely (ppFEV1 ≤P25 at ETI initiation), which identified an association of younger age with higher ppFEV1 gains at 3 and at 12 months after initiation of ETI therapy.To the best of our knowledge, our data are the first to identify larger lung function improvements toward CFTR modulator therapy in adolescents , and adoPrevious studies also indicated a more favorable response profile of children compared to adults with regard to ppFEV1. Earlier introduction of IVA or LUM/IVA led to better preservation of lung function, a larger impact on PEx , and a hOur analyses on improvements of ppFEV1 in those pwCF with severe lung disease are in a similar range as previous data by Unlike Muilwijk et al., our data failed to identify that adolescents or children show a more favorable BMI trajectory than adults . Our datOur analyses failed to identify the effects of gender on absolute values of ppFEV1 or BMI or ppFEV1 or BMI gains attained through ETI therapy, where female subjects have been shown to respond more favorably toward IVA with regard to annualized pulmonary exacerbation (PEx) rates and respond to LUM/IVA with larger BMI gains . Yet, thp = 0.001, Interestingly, the models we built also failed to indicate an effect of prior CFTR modulator therapy on absolute ppFEV1 or BMI, or ppFEV1 or BMI gains. The PROMISE Study Group showed that the highest average changes in ppFEV1 were in those pwCF previously using no modulator or a two-drug combination . Our finOur analysis has important limitations, particularly due to the heterogeneity of our subpopulations. In that line, our study cohorts differ significantly in the proportion of previous CFTR modulator therapy prior to ETI initiation (adolescents 19.5% vs. adults 50% vs. children 6–11 years 52%). Our smaller cohort with limited follow-up data may be underpowered to produce conclusive results, particularly due to unaccounted effects of previous CFTR modulator therapy. On one hand, responses to CFTR modulator therapy may be more favorable in pwCF who had had prior therapy compared to those without because the latter group may have developed irreversible changes in the intervening time which preclude further positive responses. On the other hand, improvements in response to prior CFTR modulator therapy might eventually provide a “ceiling” effect where further improvements in either ppFEV1 or BMI cannot be observed anymore. Yet, children and adolescents, with the worst lung function (≤P25) comprised the highest proportion of previous CFTR modulator therapy  in a comparable range to adults (48.6%). Regression analysis in this cohort with regard to the impact of previous modulator therapy did reveal significant influences of this variable.In addition, our age sub-groups differ in the proportion of deltaF508 homozygosity, which might impact the disease severity. Interestingly, our focus sub-group (spirometry ≤P25) contains a comparable proportion of deltaF508 homozygosity across all age groups (60%–66%), which improves the comparability of these subpopulations. Along this line, other publications also failed to identify the effects of genotype–phenotype correlations, albeit also at small cohort sizes . Still, Clinical data concerning longitudinal evolvement of pulmonary function and anthropometry, supporting age-dependent effects, are yet lacking for ETI. Such data are of particular importance in view of the large number of young patients, which became and will still become eligible for ETI. Due to previous diagnostic and therapeutic improvements, this patient population will initiate CFTR modulator therapy in an unprecedented state of health. This, combined with their young age, will render the monitoring of clinical changes in response to CFTR modulator therapy, particularly challenging. Data, as we present here, which support an association of the magnitude of response toward ETI and younger age, might lend credibility to a comprehensive approach toward the initiation of ETI at the earliest age possible, as well as advocating treatment continuation even in cases, where the subjective or objective benefits might not be immediately observable due to low functional impairment at ETI initiation.In light of the first study demonstrating long-term lung function stability in those individuals treated with ETI , the ear


We propose RabbitKSSD, a high-speed genome distance estimation tool. Specifically, we leverage load-balanced task partitioning, fast I/O, efficient intermediate result accesses, and high-performance data structures to improve overall efficiency. Our performance evaluation demonstrates that RabbitKSSD achieves speedups ranging from 5.7× to 19.8× over Kssd for the time-consuming sketch generation and distance computation on commonly used workstations. In addition, it significantly outperforms Mash, BinDash, and Dashing2. Moreover, RabbitKSSD can efficiently perform all-vs-all distance computation for all RefSeq complete bacterial genomes (455 GB in FASTA format) in just 2 min on a 64-core workstation.https://github.com/RabbitBio/RabbitKSSD.RabbitKSSD is available at  However, due to sub-optimal implementations, they usually cannot take full advantage of the resources of modern multi-core platforms. This establishes the need of a highly optimized genome similarity estimation tool on commonly used CPUs. Even though Kssd features algorithms with low computational complexity, it exhibits limited robustness and sub-optimal performance. Furthermore, when applied to large-scale genomes, Kssd requires a large amount of compute and storage resources which motivates the need for more efficient software tools.The mentioned applications provide a series of function modules for genome analysis, such as We propose RabbitKSSD, a highly optimized genome distance estimation tool featuring load-balanced task partitioning, fast I/O, efficient intermediate result accesses, and high-performance data structures. Our evaluation shows that RabbitKSSD gains speedups of at least 5.7× and 16.5× on the most time-consuming kernels of sketch generation and distance computation compared to Kssd on typical workstations. Furthermore, it clearly outperforms Mash, BinDash, and Dashing2.RabbitKSSD adopts the Kssd algorithm for estimating the similarities between genomes  and a query dataset (n genomes), Kssd maintains an intersection matrix of size of indexed dictionary into multiple sub-dictionaries, as shown in Mash distance and AAF distance are based on computing set intersections of sketch pairs. Compared to the frequently used Mash, Kssd is more efficient to compute pairwise intersections , BinDash (v.0.2.1), Dashing2 (v.2.1.14), and Kssd (v.2.21). Experiments are conducted on two Linux workstations:We have measured the performance of these tools using the NCBI RefSeq bacteria genomes, which consist of 455 GB in FASTA format , 5.2× (3.5×), 50.8× (42.0×), and 19.8× (16.5×) compared to Mash, BinDash, Dashing2, and Kssd on W1 (W2). RabbitKSSD achieves overall speedups of 17.5× (15.1×), 2.5× (2.8×), 7.4× (7.5×), and 7.5× (7.3×) compared to Mash, BinDash, Dashing2, and Kssd on W1 (W2). Apart from Dashing2, the other tools have similar memory footprints for peak memory usage. Instead of mapping the intersection matrix to hard disk in Kssd, RabbitKSSD splits the intersection matrix into lines and preserves these lines in memory. Thus, the memory footprint of RabbitKSSD is slightly higher than Kssd. Moreover, RabbitKSSD exhibits a higher memory footprint compared to BinDash, as it is required to store the unified indexed dictionary in memory for efficient retrieval during the computation of pairwise distances.The output of RabbitKSSD is identical to Kssd for all operations. For the sketch operation, RabbitKSSD gains speedups of 2.3× (2.1×), 2.2× (2.7×), 1.5× (1.6×), and 5.8× (5.7×) compared to Mash, BinDash, Dashing2, and Kssd on W1 (W2). For the I/O-intensive sketch operation, RabbitKSSD can reach the peak I/O speed of the fast SSD with multiple threads. For all-vs-all distance computation, using the default sketch size, same Furthermore, for the multi-threaded sketch generation and distance computation, RabbitKSSD achieves significantly better thread scalability than Kssd (see In conclusion, RabbitKSSD is a useful and efficient tool for comparing large-scale genome datasets on modern multi-core platforms.btad695_Supplementary_DataClick here for additional data file.