Using this CsPbI2.5Br0.5/FCQDs graded heterojunction in conjunction with low-temperature-processed carbon electrode, a power conversion efficiency of 13.53% for 1 cm2 all-inorganic perovskite solar cell can be achieved at AM 1.5G solar irradiation. To the best of our knowledge, this is one of the highest efficiency reported for carbon electrode based all-inorganic perovskite solar cells so far, and the first report of 1 cm2 carbon counter electrode based inorganic perovskite solar cell with PCE exceeding 13%. Moreover, the inorganic perovskite/carbon quantum dot graded heterojunction photovoltaics maintained over 90% of their initial efficiency after thermal aging at 85° for 1056 hours. This conception of constructing inorganic perovskite/FCQDs graded heterojunction offers a feasible pathway to develop efficient and stable photovoltaics for scale-up and practical applications.This work reports on a compositionally graded heterojunction for photovoltaic application by cooperating fluorine-doped carbon quantum dots (FCQDs in short) into the CsPbI Howevlization –8. There2/CsPbIBr2/carbon has been reported by Zhu et al., which achieved a PCE over 9% and an open-circuit voltage (VOC) of 1.245 V [2/Al2O3/NiO/carbon and inorganic perovskite CsPb0.98Mg0.02I2Br [N,N-di-4-methoxyphenylamino)-9,9′-spiro-bifluorene (spiro-OMeTAD) as the hole-transport layer (HTL) and Au as the counter electrode [In this regard, all-inorganic perovskite solar cells (PSCs) with carbon counter electrodes are thus developed in view of the structural stability and cost-effectiveness, and have become research interests arising from great prospect toward commercialization –14. For  1.245 V . Previou0.02I2Br . The ino0.02I2Br –18. The 0.02I2Br . Moreove0.02I2Br . Unfortulectrode , 21.VOC loss , as well as low fill factor (FF) for carbon electrode based PSCs [Ω/□) with large-sized particles of high porosity, indeed, minimization of the interfacial recombination kinetics gives a good shot of enhancing VOC along with FF. Therefore, in aim to enhance the photovoltaic performance of carbon electrode based all-inorganic PSCs, it is of fundamental importance to overcome the limits through the formation of intimate contact at the perovskite/carbon electrode interface and tailoring the interfacial energy-band alignment.The interface between perovskite layer and carbon electrode is crucial to the photovoltaic performance of carbon electrode based PSCs. A poor interfacial contact as well as mismatched energy level alignment between perovskite layer and carbon electrode significantly induces undesirable charge recombination –25. Thissed PSCs –14. Cons3-PTAA gradient heterojunction also achieved a PCE of 13.0% in active area of 1 cm2 [Graded-heterojunction (GHJ) engineering can be an effective method to facilitate the separation of photo-generated charge carriers and their transport in photovoltaic devices –30. For of 1 cm2 . The gra2.5Br0.5 perovskite via the antisolvent method. Because of the excellent optoelectronic properties together with high chemical stability, carbon quantum dots have shown significant potential as charge transport material or surface passivating agent in photovoltaic devices [2.5Br0.5 perovskite layer before perovskite crystallization, forming a perovskite/FCQDs graded heterojunction with increasing FCQDs percentage along the vertical direction. The results indicate that the FCQDs interact strongly with CsPbI2.5Br0.5 perovskite and efficiently passivate the uncoordinated Pb ions defects. Moreover, the CsPbI2.5Br0.5/FCQDs graded heterojunction selectively promotes photo-generated charge separation and extraction, resulting in improved photovoltaic performance. The CsPbI2.5Br0.5/FCQD-graded heterojunction in conjunction with low-temperature-processed carbon electrode achieved an impressive power conversion efficiency of 13.53% in a 1 cm2 device at AM 1.5G solar irradiation. More importantly, the solar cells based on CsPbI2.5Br0.5/FCQDs GHJ in combination with low-temperature processed carbon electrode exhibited excellent long-term stability. The unencapsulated devices maintained over 90% of their initial efficiency under thermal aging at 85°С for 1056 h.This work for the first time reports on compositionally graded heterojunction for photovoltaic application by injecting fluorine-doped carbon quantum dots (FCQDs in short) into inorganic CsPbI devices –34. In t2.5Br0.5/FCQDs graded heterojunction was fabricated by injecting FCQDs into inorganic CsPbI2.5Br0.5 perovskite layer via antisolvent method during perovskite precursor spin-coating process as illustrated in 2.5Br0.5 perovskite film before crystallization, while most of FCQDs maintain on perovskite layer surface, forming a CsPbI2.5Br0.5/FCQDs graded heterojunction structure with increasing FCQDs percentage along the vertical direction. This GHJ conception was verified by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) characterization as shown in 2.5Br0.5/FCQDs GHJ thin film by using Bi3+ ion beam, the SIMS characterization detects the signals of Pb, I, Br, and C in negative mass detection mode, in which C was used as an indicator of FCQDs. Obviously, the C depth profile (blue line) shows a steep decline at the perovskite/FCQDs GHJ film surface and negligible counting amount in the bulk. The decrease of carbon content along the vertical direction verifies a graded distribution of FCQDs in the CsPbI2.5Br0.5 perovskite absorption layer.The CsPbI2.5Br0.5 and FCQDs was estimated by ultraviolet photoelectron spectroscopy (UPS) and UV-visible absorption spectroscopy characterization  of CsPbI2.5Br0.5 and FCQDs was calculated to be −4.36 and −4.74 eV, respectively  of CsPbI2.5Br0.5 and FCQDs was further evaluated to be −5.61 eV and −5.37 eV, respectively. By taking into account of the valance band maximum, the conduction band minimum, and the Fermi level of both CsPbI2.5Br0.5 and FCQDs, upon contact, electron is depleted from CsPbI2.5Br0.5 to FCQDs, causing an upward band bending at the CsPbI2.5Br0.5/FCQDs interface. Base on this understanding, we built up an energy-level diagram for the perovskite/FCQDs GHJ in 2.5Br0.5 at the heterojunction interface. In addition to concentration gradient, this kind of band bending supplies additional driving force for photogenerated hole extraction and restrains electron flowing back.The interfacial band alignment around CsPbIn Figure a-S2c. Th7/2) and Pb(4f5/2) at 138.2 eV and 143.05 eV for CsPbI2.5Br0.5 sample positively shift to higher values by 0.015 eV for CsPbI2.5Br0.5/FCQDs GHJ sample. This is usually caused with the existence of additional negative charges surrounding Pb ions [2.5Br0.5 thin film and CsPbI2.5Br0.5/FCQDs GHJ thin films on glass substrates. By formation of graded heterojunction, the PL emission peak changed from ~1.81 eV to ~1.82 eV for the CsPbI2.5Br0.5 perovskite thin film. A blue-shifted PL emission peak can be correlated with filling of the trap states on CsPbI2.5Br0.5 layers' surface or along the grain boundaries by injection of FCQDs [2.5Br0.5/FCQDs GHJ thin film as a result of charge transfer from CsPbI2.5Br0.5 to FCQDs. This was further confirmed by time-resolved PL decay characterization, 2.5Br0.5/FCQDs GHJ sample demonstrates substantial photogenerated charge carriers transfer from CsPbI2.5Br0.5 to FCQDs.Figure  Pb ions –38. In t Pb ions . This co Pb ions . Figure of FCQDs . Moreove2.5Br0.5/FCQDs GHJ on charge transfer, we investigated the electrical potential distribution across the GHJ structure by performing scanning Kevin probe force microscopy (KPFM) characterization on cross-section of the FTO/TiO2/CsPbI2.5Br0.5/Au and the FTO/TiO2/CsPbI2.5Br0.5/FCQDs GHJ/Au samples in dark condition as shown in Figure 2/CsPbI2.5Br0.5/Au sample which exhibited a relatively constant potential distribution across the perovskite absorption layer, a potential drop of about 120 mV was observed at the perovskite/FCQDs graded heterojunction area at −1.0 V bias voltage in the FTO/TiO2/CsPbI2.5Br0.5/FCQDs GHJ/Au sample. This obvious potential drop can be caused by charge diffusion motion at the perovskite/FCQDs interface, which also demonstrates the existence of depletion region at the perovskite/FCQDs heterojunction interface. The electric field distribution profiles can be determined as shown in Figures To gain more insight about the construction of CsPbI2.5Br0.5/FCQDs GHJ PSCs using low-temperature processed carbon electrode as current collector. With the merit of low-temperature large-area processability and low manufacture cost, the carbon electrode based CsPbI2.5Br0.5/FCQDs GHJ PSCs can be easily scaled up for practical applications. 2.5Br0.5/FCQDs film can be clearly observed. J − V) measurement curves of the optimized 1.0 cm2 PSC devices. We observed that the PSC device (device B) based on perovskite/FCQDs GHJ exhibited improved photovoltaic performance. The champion device showed a VOC of 1.12 V, a JSC of 16.87 mA cm−2, a FF of 71.6%, yielding an impressive PCE of 13.53%. This is one of the highest PCE among carbon electrode-based inorganic PSCs reported so far. In contrast, the control device (device A) showed a total PCE of 10.70%, with a VOC of 1.05 V, a JSC of 15.97 mA cm−2, and a FF of 63.8%. VOC loss for currently reported PSCs  as light absorber and carbon counter electrode as current collector. Apparently, our present fabricated CsPbI2.5Br0.5/FCQDs GHJ device exhibited both high PCE and low VOC loss. This result substantiates the promising potential for constructing perovskite/FCQDs GHJ toward highly efficient PSCs. To the most of our knowledge, this is the first report of 1 cm2 carbon counter electrode based inorganic PSC with PCE exceeding 13%. JSC calculated from the EQE spectra for devices A and B were estimated to be 15.40 and 16.19 mA cm−2, respectively.We further fabricated CsPbI−2 illumination. Two semicircles were observed in the frequency analysis. The first arc corresponds to charge transport processes in the charge transport layers or at the charge transport layer/counter-electrode interfaces in the high-frequency region, i.e., fast charge transport processes. The second arc can be assigned to charge recombination processes at the perovskite layer/charge selective contact interfaces in the intermediate frequency region, i.e., relative slow charge transport processes [RCE) at the perovskite layer/counter-electrode interface, which indicates faster transport of holes from the perovskite absorber to the carbon electrode. RCT) as a function of bias voltage. Apparently, device B exhibits larger RCT under a series of bias voltage, indicating a slower interfacial charge recombination process. Moreover, a relatively smaller capacitance was calculated for device B from the response in the intermediate frequency region  measurements were conducted to understand the effect of perovskite/FCQDs GHJ on electronic processes at the perovskite/carbon electrode interface. The measurements were performed by connecting the FTO glass as working electrode and carbon electrode as counter electrode and reference electrode –52.  Figrocesses . We fittn Figure , implyinτre) at a different open-circuit voltage (or quasi-Fermi level) can be obtained in the model of TPV. Similarly, the apparent transport lifetime (τtr) is collected at short circuit conditions in TPC measurements. The transport/recombination kinetics in PSCs are frequently modeled with biphasic decay  for the devices by fitting the TPC curves. Notably, both electron and hole transport lifetimes of device B are significantly shorter than that of device A. At an identical voltage of ~0.9 V, the transport lifetime of electron  of device B is approximately 6 μs, being shorter than that of device A (~8 μs). Similarly, the transport lifetime of hole  of device B is approximately 1.5 μs, being shorter than that of device A (~2.6 μs). This result can be attributed to the CsPbI2.5Br0.5/FCQDs GHJ, which introduces additional driving force for hole transfer, therefore an enhanced charge carrier transport. This result also indicates an imbalanced charge transport in both devices. A similar behavior has been found in the TPV measurements (τre) of electron and hole compared to device A. At an identical voltage of ~0.9 V, the recombination lifetime of electron  of device B is approximately 31.5 μs, being longer than that of device A (~23 μs). Similarly, the recombination lifetime of the hole  of device B is approximately 7 μs, being longer than that of device A (~2.9 μs). A long interfacial recombination lifetime guarantees efficient charge collection and thus enhancing the device output photovoltage. The diffusion length (L) of charge carriers in CsPbI2.5Br0.5 PSCs was further evaluated by the ratio of recombination lifetime and transport lifetime, as given by the expression L = (D × τre)1/2, where D is the charge carrier diffusion coefficient obtained from charge transport time τtr. As shown in L). At an identical voltage of ~0.9 V, the diffusion length of electron (Le) of device B is approximately 1.46 μm, being longer than that of device A (~1.06 μm). The diffusion length of the hole (Lh) of device B is approximately 617 nm, being longer than that of device A (~303 nm). In comparison with previous report on CsPbI2Br-based inorganic PSC showing diffusion length of 463 nm for electrons and 202 nm for holes, in this study, the CsPbI2.5Br0.5/FCQDs GHJ device exhibited much longer diffusion length for both electrons and holes [To examine the effect of perovskite/FCQDs GHJ on dynamics of charge carrier transfer and recombination, the PSC devices were further characterized with transient photocurrent/photovoltage decay (TPC/TPV) measurements at various bias light intensities Figure . The wory Figure , correspurements , showingnd holes . Especia2.5Br0.5 perovskite/FCQDs GHJ PSCs, the unencapsulated CsPbI2.5Br0.5 devices (control) and CsPbI2.5Br0.5/FCQDs GHJ devices were measured under 85°C thermal-aging condition in argon-filled glove box. Herein, 85°C was selected for thermal-aging according to the standard IEC 61215-1 : 2016 [2.5Br0.5 devices (control) maintained 84.3% of the initial efficiency, while the CsPbI2.5Br0.5/FCQDs GHJ device maintained 90.7% of the initial efficiency. In contrast, the PSC device using spiro-OMeTAD as HTL and Au as counter-electrode almost lose 100% of the initial efficiency within 200 hours. The considerable improvement in long-term thermal stability of the carbon electrode-based CsPbI2.5Br0.5/FCQD GHJ devices can be attributed to the elimination of organic HTL and metal-electrode, as well as the robust shielding effect of FCQDs. These results demonstrate the significance of constructing carbon electrode-based inorganic perovskite/FCQDs GHJ PSCs for achieving long-term stability and provide important direction to realize low cost and highly stable inorganic PSCs toward commercialization.Long-term stability is another momentous metrics for the practical application of PSCs . To esti1 : 2016 , and the1 : 2016 , 59. The2.5Br0.5. This CsPbI2.5Br0.5/FCQDs GHJ can effectively facilitate the separation of photo-generated charge carriers and their transport in photovoltaic devices. Using this novel inorganic perovskite/FCQDs graded heterojunction in conjunction with low-temperature processed carbon electrode, a PCE of 13.53% can be achieved over 1 cm2 device. Moreover, the carbon electrode based CsPbI2.5Br0.5/FCQDs GHJ PSCs exhibited excellent long-term stability, maintaining 90.7% of the initial efficiency under 85°С thermal aging for 1056 hours. This inorganic perovskite/FCQDs graded heterojunction offers a visible pathway to solve charge extraction obstacle in large-area carbon electrode based inorganic PSCs and provides an important direction to realize low-cost and highly stable inorganic PSCs toward commercialization.In summary, we first present a compositionally graded heterojunction structure for photovoltaic application by injecting fluorine-doped carbon quantum dots into inorganic perovskite CsPbISynthesis of fluorine-doped carbon quantum dots (FCQDs): a one-step hydrothermal method was adapted to prepare FCQDs . Briefly2 layer was prepared by spray pyrolysis deposition of titanium isopropoxide ethyl alcohol solution, followed by annealing at 100°C for 10 min and 450°C for 30 min. The mesoporous TiO2 layer was prepared by spin-coating of 18 nm particle size paste (Dyesol 18 NR-D) diluted in ethanol at 3000 for rpm 30 s, then sintered at 100°C for 10 min and 450°C for 1 hour. The 0.8 M CsPbI2.5Br0.5 perovskite precursor solution was made by dissolving CsI, CsBr, and PbI2 (molar ratio 1 : 1 : 2) in a mixture of DMF and DMSO  and stirred overnight. For the graded heterojunction device, 2 mg/mL FCQDs were added into chlorobenzene antisolvent and stirred overnight. The perovskite solution was spin-coated onto FTO/TiO2 substrates at 2000 rpm for 10 s and 6000 rpm 30 s, and chlorobenzene with or without FCQDs was dropped onto the film at the last 23 s during the second step. Subsequently, the spin-coated perovskite films were dried for 5 min, then thermally annealed at 70°C for 3 min and 300°C for 10 min. Finally, the carbon paste was coated by the doctor-blade method and annealed at 100°C for 30 min [PSC devices fabrication: the compact TiOr 30 min .Other experimental details and characterizations are shown in the Supporting Information.


Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against Mycobacterium tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and antibiofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10−8/CFU and mapped to gyrA and gyrB encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild-type enzyme but not the P4C-resistant mutant. P4C-resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide-resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in recA promoter-based DNA damage reporter strains showed induction of recA promoter activity in the wild type but not in the P4C-resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.New, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens,  Mycobacterium abscessus and Mycobacterium avium complexes  are increasing –3. NTM l–omplexes . Althoug factors . In addi factors . The cur factors , 8. M. a disease , 10. Itssfactory , 12. Conpatients , 14. Mosregimens . Thus, n disease –18.M. tuberculosis were identified through successful whole-cell screening campaigns, leading to a reenergized tuberculosis drug discovery and development pipeline (https://www.newtbdrugs.org/). We and others have shown that focused libraries of compounds active against M. tuberculosis provide an attractive source of hits active against NTM (–https://www.mmv.org/mmv-open/pathogen-box), we identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit active against our M. abscessus screening strain M. abscessus subsp. abscessus Bamboo . By substituting the chlorine in position 4 of the phenyl moiety of 844 with a trifluoromethyl group , we iden to 6 μM .M. abscessus subsp. abscessus Bamboo treated with 844 in broth culture, suggesting bactericidal activity of the compound , harboring a D91N missense mutation in gyrA associated with high-level P4C resistance (M. abscessus subsp. abscessus ATCC 19977 wild-type (wt) gyrAB, which partially restored sensitivity to both 844 and 844-TFM  of 844-TFM (1.9 μM) was 2.4-fold lower than the IC50 of the parental 844 (4.6 μM) (M. abscessus DNA gyrase harboring the resistance mutation D91N in gyrA (M. abscessus subsp. abscessus ATCC 19977 844-TFMr-1)  . To conf-TFMr-1) ; Fig. 3.-TFMr-1) .M. abscessus DNA gyrase, an essential and clinically validated target in mycobacteria  liabilities of 884-TMF, we determined its concentration-time profile in mice and measured basic PK properties in established  to 0.2% . To deteK assays . 844-TFMK assays , nearly K assays , predictK assays . HoweverK assays . IdentifK assays . Taken tin vitro susceptibilities due to (unknown) intrinsic resistance mechanisms limit the therapeutic utility of this drug class against M. abscessus  T28 sequevar  T28 sequevar  deletion sequevar  supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% albumin-dextrose-catalase. 7H10 agar  was used as solid medium.844 was obtained from the Medicines for Malaria Venture’s (Geneva) compound archive. 844-TFM was synthesized as described below. Moxifloxacin  and clarithromycin  were purchased from Sigma-Aldrich, USA. SPR719 (HY-12930) was purchased from MedChemExpress LLC, USA. MXF, SPR719, and CLR were dissolved in 100% dimethyl sulfoxide (DMSO)  at 10 mM. 844 and 844-TFM were dissolved in ethanol  at 5 mM and 10 mM, respectively. All compounds were stored in aliquots at −20°C until use.N--4-piperidinecarboxamide was prepared according to the literature (1H and 13C nuclear magnetic resonance (NMR) spectra were recorded at room temperature on an Agilent Technologies VNMRS 500 NMR spectrometer. The residual solvent signals of methanol-d4  were used to reference the spectra . The mass spectrum was recorded on a Q Exactive Plus Orbitrap mass spectrometer  using methanol as solvent.Starting materials were purchased and used as received. Solvents were of reagent grade and distilled before use. terature , 46. TheN--4-piperidinecarboxamide , triethylamine , and 1-(2-bromoethyl)-4-(trifluoromethyl)benzene  in dimethylformamide (DMF) (5 ml) was stirred at 50°C for 16 h. The reaction was quenched by the addition of water (20 ml). The mixture was extracted with ethyl acetate (3 × 10 ml), and the combined organic phases were washed with water (10 ml) and brine (10 ml). The solution was dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was chromatographed on silica gel with methanol/ethyl acetate (0 to 5%) to afford the product as a white solid ; M.p. 433-435 K; 1H NMR  δ 8.61 , 8.50 , 8.19 , 7.58 , 7.43 , 7.26 , 4.16 , 3.14 , 2.96 to 2.91 , 2.74 to 2.66 , 2.27 , 2.09 , 1.93  ; 13C NMR  δ 174.7, 161.8, 148.3, 144.7, 144.7, 140.4, 140.3, 139.2, 132.0, 129.0, 128.2, 128.0, 125.5, 124.9 , 124.9, 124.9, 124.8, 117.1, 110.8, 59.6, 53.3, 52.4, 43.5, 32.4, 28.1, 19.3 ; electrospray ionization-high-resolution mass spectrometry (ESI-HRMS): m/z [M+H]+ calculated for C24H26F3N4O 459.2007, found 459.1995.A mixture of M. abscessus cultures  were adjusted to a density of OD600 = 0.01 in Middlebrook 7H9 broth . One hundred microliters of the bacterial suspension was seeded onto the 96-well plates containing 100 μl of the serially diluted compounds to give a final volume of 200 μl in each well with the final OD600 of 0.005. The plates were sealed with parafilm, placed onto wet paper towels in a lock-lock box and incubated for 3 days at 37°C with orbital shaking at 90 rpm. Absorbance at 600 nm was measured using a TECAN Infinite Pro 200 plate reader after resuspension. Absorbance values at day 3 were subtracted from the day 0 readout. Percent growth inhibition was calculated by dividing the absolute absorbance value of treated cells with untreated control and multiplying by 100. CLR was included in all MIC experiments as a positive control to monitor assay reproducibility. MIC was defined as 90% of growth inhibition relative to untreated controls.MIC was determined using the broth microdilution method in 96-well plates as described previously . BrieflyM. abscessus subsp. abscessus ATCC 19977 cultures (OD600 = 0.4 to 0.8) were diluted to a final OD600 of 0.005, and 1 ml of the culture suspension was exposed to 1×, 4×, and 8× MIC of 844 (MIC = 8 μM), 844-TFM (MIC = 1.5 μM), MXF (MIC = 3 μM), SPR719 (MIC = 0.6 μM), and CLR (MIC = 2 μM) in 14-ml vented, round-bottom tubes  and incubated at 37°C on an orbital shaker at 200 rpm. At 0, 2, and 3 days, tubes were vortexed, and 20 μl of the cultures was transferred to round-bottom 96-well plates containing 180 μl of phosphate-buffered saline (PBS)  with 0.025% Tween 80 (PBS/Tween 80) for 10-fold serial dilution. To prevent compound carryover effects, we plated out serially diluted samples onto 7H10 agar supplemented with 0.4% activated charcoal  for all time points as described . Bacteria were resuspended in ‘7H9’ to an OD600 of 0.0125, and 150 μl was dispensed into the wells of Innovotech 96-well plates. Pegs  were inserted into the bacterial suspensions in the wells, and the cultures were grown 24 h with shaking at 110 rpm at 37°C to allow attachment and growth of the bacteria on the surface of the pegs. Then, the lids with the pegs were transferred to a new multititer plate containing 150 μl fresh ‘7H9’ broth   as described previously  with minme zero)  with no M. abscessus subsp. abscessus Bamboo and M. abscessus subsp. abscessus ATCC 19977 as described previously . The plates were sealed with parafilm and incubated for 7 days at 37°C. Apparent resistant colonies were purified and confirmed by restreaking on 7H10 agar containing the same concentration used for selection of resistant colonies. Genomic DNA was extracted as described previously using the phenol-chloroform method (gyrA (MAB_0019) and gyrB (MAB_0006) by whole-genome sequencing were confirmed by Sanger sequencing  using primers for gyrA  and gyrB .Spontaneous resistant mutants against 844 and 844-TFM were selected using eviously . Brieflym method . Whole-gM. abscessus subsp. abscessus ATCC 19977 844-TFMr-1 strain harboring a D91N allele of gyrA  using primers Fw-gyrBA  5′-aagcttCATCATCATCATCATCACGCTGCCCAGAAGAAGAGTGgcg-3′ and Rv-gyrBA  5′-aagcttTTACTCGCCTGCGGTCGTGgcg-3′ (lowercase sequences indicate added sequences with restriction enzyme recognition sites underlined) and Phusion high-fidelity DNA polymerase . To allow in-frame cloning of the gyrBA coding sequences to the N-terminal hsp60 coding sequence via pMV262’s HindIII site, pMV262’s DraI-HindIII segment  was replaced by a shortened DraI-HindIII fragment amplified using the primers Fw-hsp60  5′-tttaaaTCTAGAGGTGACCACAACGcgc-3′ and Rv-hsp60  5′-aagcttCGCAATTGTCTTGGCCATTGCgcg-3′) and pMV262 as template. The HindIII-digested gyrBA insert was ligated into HindIII-digested modified pMV262 plasmid and transformed into Escherichia coli DH5α. pMV262-phsp60-gyrBA harboring transformants was identified by PCR, and the plasmid was electroporated M. abscessus subsp. abscessus ATCC 19977 844-TFMr-1 as described (hsp60-gyrBA. M. abscessus subsp. abscessus ATCC 19977 844-TFMr-1 harboring pMV262-phsp60-gyrBA or modified pMV262-phsp60 plasmid without gyrBA insert (pMV262-empty) were treated with 844 or 844-TFM or the negative control CLR at 1× MIC in 14-ml vented, round-bottom tubes  and incubated at 37°C on an orbital shaker at 200 rpm. After 2 days of incubation, tubes were vortexed and 200 μl of the samples from each tube transferred into a clear bottom 96-well plate for absorbance measurement at 600 nm using a TECAN Infinite Pro 200 plate. A two-way ANOVA with Sidak's multiple comparison test was performed using GraphPad Prism 8 software to compare the two groups.To complement the P4C-resistant  of gyrA , wild-tyo pMV262  and expreviously . The codescribed . TransfoM. abscessus DNA gyrase gyrA and gyrB , we adapted the strategy previously used for expression of recombinant M. tuberculosis DNA gyrase  5′-gctagcATGACTGACACAACGCTGCCCCCCgcagca-3′, Rv-gyrA  5′-aagcttTTACTCGCCTGCGGTCGTGCCGGCtgctgc-3′, Fw-gyrB  5′-catatgGCTGCCCAGAAGAAGAGTGCCAAGgcagca-3′, and Rv-gyrB  5′-gctagcTACGTCGAGGAAGCGCACGTCCtgctgc-3′. The fusion GyrB and GyrA with an alanine-serine linker was cloned into pET-28a(+) containing an N-terminal hexahistidine tag using the restriction enzymes NdeI (R0111S), NheI (R3131S), and HindIII (R3104M) from New England BioLabs, resulting in the plasmid pET28a+gyrBA wt. For expression of recombinant DNA gyrase harboring a D91N substitution in GyrA, the analogous plasmid pET-28a+gyrBAD91N was generated via gene synthesis and cloning using Genewiz’s gene synthesis service.To clone and express recombinant A gyrase . The codE. coli BL21(DE3) pLysE by growing the cells at 37°C until early log phase and then treating the cultures with 1 mM isopropyl-β-d-thiogalactopyranoside for 3 h at 30°C . Cells were harvested by centrifugation and resuspended in buffer A500  with protease inhibitor cocktail, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, bestatin, E64, leupeptin, and pepstatin A . For protein purification, cells were sonicated and centrifuged , and the clarified lysate was passed over an Ni gravity column  preequilibrated with the buffer A500. The column was washed with 10 column volumes of buffer A500. The protein was eluted with buffer B500 , and the fractions were pooled and dialyzed overnight at 4°C against buffer A500. For the biochemical assays, the proteins were concentrated using Pierce protein concentrator PES , aliquoted, and stored at −20°C.Wild type and GyrA D91N mutant DNA gyrase were expressed and purified as described . ProteinM. abscessus instead of the recombinant M. tuberculosis DNA gyrase supplied by the kit. The assay was carried out in a 30-μl final volume, containing 50 mM HEPES KOH (pH 7.9), 6 mM magnesium acetate, 4 mM dithiothreitol (DTT), 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine, 0.05 mg/ml bovine serum albumin, 40 nM either wild-type or GyrA D91N mutant DNA gyrase, and 0.5 μl (1 μg/μl) of relaxed pBR322 DNA. The reaction mixture was incubated for 30 min at 37°C, and then stopped by adding 30 μl of chloroform/isoamyl alcohol (24:1) followed by 30 μl of STEB  solution. The tubes were briefly vortexed and centrifuged for 1 min, and 20 μl of the aqueous (upper blue) phase was loaded onto a 1% (wt/vol) agarose gel. The gel was run for 2 h at 75 V and stained with 1 μg/ml ethidium bromide in water. After destaining with water for 10 min, images were taken and analyzed using Invitrogen iBright FL1000 imaging system. To determine IC50 values, the intensity of the bands was measured and compared to the drug-free reaction using iBright analysis software. The IC50 values were determined using nonregression model fit of GraphPad Prism 8.0.1 software.The mycobacterial DNA gyrase inhibition assay was performed using the kit from Inspiralis Limited  as described by the suppliers using the generated recombinant DNA gyrases from M. abscessus cultures harboring a LuxCDABE reporter expressed under the control of the recA promoter was measured. A recA-LuxCDABE reporter plasmid was constructed by replacing the NotI-EcoRI hsp60 promoter fragment of pMV306hsp60-LUX  5′-gcggccgcGTTGGGGGAACCGCGTTACgc-3′ and Rv-PrecA  5′-gaattcGGTGTTCTCCGTTTCGTCGccg-3′ using Phusion high-fidelity DNA polymerase . The resulting amplicon was digested with NotI  and EcoRI , gel purified , and ligated into NotI-EcoRI-digested pMV306hsp60-LUX, resulting in the reporter plasmid pMV306recA-LUX  were adjusted to an OD600 of 0.1 and treated with various concentrations of drugs. Luminescence intensity was measured using a TECAN Infinite Pro 200 plate reader at 250 ms integration time with automatic attenuation at time zero and after 4 h at 37°C. Data analysis was performed using GraphPad Prism 8 software after subtracting the luminescence at time zero.To assess whether compounds cause DNA damage, luminescence of sp60-LUX   permeability, and stability in the mouse liver microsome assay were performed by BioDuro  according to standard protocols.N,N-dimethylacetamide (DMA)/95% Milli-Q water vehicle. Blood samples were serially collected via the tail snip from each individual mouse at 0.017, 0.25, 1, 3, 5, and 8 h postdose following i.v. dosing and at 0.5, 1, 3, and 5 h postdose following p.o. and s.c. dosing. Blood (50 μl) was collected in capillary Microvette K2EDTA tubes  and kept on ice prior to centrifugation at 1,500 × g for 5 min. The supernatant (plasma) was transferred into a 96-well plate and stored at −80°C.All animal experiments were approved by the Center for Discovery and Innovation, Institutional Animal Care and Use Committee, and were conducted in compliance with their guidelines. Female CD-1 mice were weighed and received a single dose of 884-TFM via the intravenous (i.v.) (5 mg/kg of body weight), oral (p.o.) (25 mg/kg), or subcutaneous (s.c.) (25 mg/kg) dosing route. The compound was formulated as a solution in 5% 2EDTA Mouse; Bioreclamation IVT). Twenty microliters of control, standard, QC, or study sample were added to 200 μl of ACN/methanol (MeOH) 50:50 protein precipitation solvent containing internal standard (10 ng/ml verapamil). Extracts were vortexed for 5 min and centrifuged at 4,000 rpm for 5 min. One hundred microliters of supernatant was transferred for HPLC-MS/MS analysis and diluted with 100 μl of Milli-Q deionized water.Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on a Sciex Applied Biosystems Qtrap 6500+ triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera 2 high-pressure liquid chromatography (HPLC) system to quantify each drug in plasma. Neat 1 mg/ml DMSO stocks for 844-TFM were serial diluted in 50:50 acetonitrile (ACN)/water to create standard curves and quality control (QC) spiking solutions. Standards and QCs were created by adding 10 μl of spiking solutions to 90 μl of drug-free plasma  using a reverse-phase gradient. Milli-Q deionized water with 0.1% formic acid was used for the aqueous mobile phase and 0.1% formic acid in ACN for the organic mobile phase. Multiple-reaction monitoring of precursor/product transitions in electrospray positive-ionization mode was used to quantify the analytes. The following multiple reaction monitoring (MRM) transitions were used for 844-TFM (459.10/133.00) and verapamil (455.4/165.2). Sample analysis was accepted if the concentrations of the quality control samples were within 20% of the nominal concentration. Data processing was performed using Analyst software .Chromatography was performed on an Agilent Zorbax SB-C2EDTA anticoagulant (Bioreclamation). Stability samples consisted of 5 μl of stock compound solution in 50:50 ACN/water and 95 μl of plasma to a final concentration of 1 μg/ml. The samples were incubated at 37°C with shaking; 10 μl of plasma was removed at each time point and combined with 100 μl of ACN/methanol 50:50 protein precipitation solvent containing internal standard (10 ng/ml verapamil). Chromatography for metabolite identification was performed the same as specified for pharmacokinetic sample analysis. Full scan total ion chromatograms (TIC) of plasma extracts were acquired using a Q Exactive high-resolution mass spectrometer (QE-HRMS) at 70,000 mass resolution. Thermo Fisher Compound Discoverer software was used to assist in identifying the 844-TFM metabolites from the QE-HRMS TIC mass spectrum. Figure S2 in the supplemental material illustrates the extracted ion chromatograms (XIC) of 844-TFM and the metabolites using 5 ppm mass accuracy before and after 12 h of incubation in mouse plasma.The plasma stability assays were carried out using plasma from female CD-1 mice, New Zealand white rabbits, rhesus monkeys, and humans containing K


T was isolated from rhizosphere soil of Bruguiear gymnorrhiza collected from the Beibu Gulf of China. Optimum growth occurred at 28 °C, pH 7.0, and under the conditions of 3–5% (w/v) NaCl. The phylogenetic comparisons of 16S rRNA gene sequences displayed that strain BGMRC 2036T was closely related to Martelella limonii NBRC109441T (96.6% sequence similarity), M. mediterranea CGMCC 1.12224T (96.5%), M. lutilitoris GH2-6T (96.5%), M. radicis BM5-7T (96.2%), and M. mangrove BM9-1T (95.9%), M. suaedae NBRC109440T (95.8%). The phylogenomic tree based on the up-to-date bacterial core gene set indicated that the strain BGMRC 2036T form a clade formed with members of the genera Martelella. The major polar lipids include phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphotidylinositol, two unidentified phospholipids, and three unidentified ninhydrin positive phospholipids. The major respiratory quinone is Q-10, which is similar to those of genera Martelella. The main cellular fatty acids are C18:1ω7c, C16:0, and C12:0 aldehyde. Genome sequencing revealed a genome size of 4.99 Mbp and a G + C content of 62.3 mol%. Pairwise comparison of the genomes of the new strain BGMRC 2036T and the three reference strains M. endophytica YC 6887T, M. mediterranea CGMCC 1.12224T, and M. mangrovi USBA-857 indicated that gANI value was lower than 81% and a digital DNA–DNA hybridization value was lower than 27%. The strain BGMRC 2036T possessed genes putatively encoding riboflavin synthesis and flavodoxin A polyphasic taxonomic study suggested that strain BGMRC 2036T represented a novel species belonging to the genus Martelella, and it was named Martelella alba sp. nov. The type strain is BGMRC 2036T (=KCTC 52121T =NBRC 111908T).Strain BGMRC 2036The online version contains supplementary material available at 10.1007/s00203-020-02178-2. Martelella of the family Aurantimonadaceae was originally described by Rivas et al. (https://lpsn.dsmz.de/genus/martelella), M. mediterranea as the type species, which were isolated from Lake Martel in Mallorca . The rhizosphere soil was stored in a sterile plastic bottle at 4 °C as soon as it was collected and then transported to the laboratory within 12 h. Soil (2 g) was added to 20 mL sterilized seawater, shaken at 37 °C for 1 h, and then diluted by tenfold. After being incubated at 28 °C for 1 week, 100 μL of the diluent was spread on modified Yeast Malt Extract -glucose anhydrous, 15.0 g agar powder, and 1 L sterilized seawater). A cream colony isolation and purification on modified ISP2 medium was designed a new strain and then stored at − 80 °C with a 20% (v/v) glycerol suspension. Strains M. suaedae NBRC109440T, M. limonii NBRC109441T, and M. mediterranea CGMCC 1.12224T, obtained, respectively, from the National Biological Resource Center, NITE (NBRC) and China General Microbiological Culture Collection Center (CGMCC), were used as references.During our investigations of microbial biodiversity in mangrove plants, strain BGMRC 2036T was performed as described by Smibert and Krieg  hydrogen peroxide (H2O2) solution  was used for the analysis of cell morphology. The presence of strain-flagella was checked by transmission electron microscopy  after 2 days of growth on modified ISP2 medium at 28 °C. Cell motility determination was realized by investigating the development process of turbidity throughout a tube using modified ISP2 semisolid medium containing 0.4% agar Leifson . Gram stnd Krieg . OxidaseT and the reference strain were harvested after cultivation on modified ISP2 medium at 28 °C for 3 days, whose polar lipids were resulted by extraction as described by Kamekura   according to the study of Meier-Kolthoff et al.  . There were 28, 13, and 25 different reactions of the 95 tested substrates (Biolog GEN III MicroPlate) between strains BGMRC 2036 and M. mediterranea CGMCC 1.12224T, M. suaedae NBRC109440T, and M. limonii NBRC109441T (Table S2). The differences of physiological and biochemical characteristic between strain BGMRC 2036T and its closely related type strains are listed in Table According to API 50CH, strain BGMRC 2036T was C18:1ω7c (48.6%). The remaining fatty acid component (> 10%) included C16:0 (22.1%), C12:0 aldehyde (14.2%), iso-C16:1, and C14:0 3-OH (13.9%), which were similar to that of M. suaedae NBRC 109440T. However, the minor fatty acids C16:1ω7c and C16:1ω6c were discovered in BGMRC 2036T and were not present in M. suaedae NBRC 109440T. The fatty acid profile of the new isolate closely resembled those of the type strains of recognized Martelella species, although some differences in their proportions were observed. The detailed fatty acid profiles of strain BGMRC 2036T and its related reference strains are shown in Table T was similar to that of the type strains of the genus Martelella, with phosphatidylmethylethanolamine and phosphatidyl inositol as the predominant components; phosphatidylglycerol, phosphatidylcholine, one unidentified phospholipid (PL3) and three unidentified ninhydrin positive phospholipids (NPL1–3) were only detected in BGMRC 2036T. Furthermore, the absence of phosphatidylethanolamine, one unidentified phospholipid (PL2), seven unidentified ninhydrin positive lipids (AL1–7) and seven unidentified polar lipids (L1–7), along with the presence of phosphatidylglycerol, phospatidyl choline, and two unidentified ninhydrin positive phospholipids (NPL2–3) in the BGMRC 2036T lipid profile helped distinguish the strain from M. mediterranea, M. suaedae, and M. limonii . Hence, from the data obtained above, strain BGMRC 2036T could clearly be differentiated from its closest phylogenetic relatives. The menaquinone was ubiquinone Q-10, which was identical to that of the Martelella genus.The major fatty acid of BGMRC 2036T was closely related to M. limonii NBRC 109441T (96.6% sequence similarity), M. mediterranea CGMCC1.12224T (96.5%), M. radicis BM5-7T (96.2%), and M. suaedae NBRC109440T (95.9%). Phylogenetic analysis based on the neighbor-joining algorithm, maximum-likelihood algorithm, and minimum-evolution methods revealed that strain BGMRC 2036T formed a clade within members of the genus Martelella related to the family Aurantimonadaceae  is a diazotroph resource in mangrove sediment, which may have relationship with the habitat of mangroves plants .O-nitrophenyl-β-d-galactopyranoside, VP test, glucose fermentation, glucose fermentation, amygdalin, and arabinose were positive. In the API ZYM, alkaline phosphatase, esterase (C4), leucine arylamidase, yaline arylamidase, acid phosphatase, naphthol-ASBI-phosphohydrolase, β-galactosidase, α-glucanase, β-glucosidase, and N-acetyl-β-glucosaminidase activities were positive. The major fatty acids of strain BGMRC 2036T were C18:1ω7c and C16:0, while ubiquinone Q-10 was found to be the predominant menaquinone. The main polar lipids included phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidyl inositol, two unidentified phospholipid (PL1 and PL3), and three unidentified ninhydrin positive phospholipid (NPL1-3). This strain type was BGMRC 2036T (=KCTC 52121T =NBRC 111908T) isolated from the rhizosphere soil of B. gymnorrhiza from the Beibu Gulf.The Gram-negative, non-motile, and rod-shaped bacteria cells are 0.3–0.4 μm in width and 0.6–1.0 μm in length. Colonies were moist, circular, smooth, white, and 0.1–0.5 mm in diameter after being maintained on modified ISP2 agar at 28 °C for 2 days. Growth occurred at 25–37 °C  with pH range 6.0–11.0 (pH 7.0) and containing 0–8.0% (w/v) NaCl (3–5%). The strain was negative for nitrate reduction, hydrolysis of gelatin, cellulose, starch, Tween 20, 40, and 80, and milk coagulation and peptonization. In the API 20E, Supplementary file1 (DOCX 2968 KB)Below is the link to the electronic supplementary material.


Morinda citrifolia) is widely dispersed throughout Southeast Asia, the Caribbean, and Australia. We previously reported that fermented Noni could alleviate atopic dermatitis (AD) by recovering Th1/Th2 immune balance and enhancing skin barrier function induced by 2,4-dinitrochlorobenzene. Noni has a high deacetylasperulosidic acid (DAA) content, whose concentration further increased in fermented noni as an iridoid constituent. This study aimed to determine the anti-AD effects and mechanisms of DAA on HaCaT, HMC-1, and EOL-1 cells. DAA inhibited the gene expression and secretion of AD-related cytokines and chemokines including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-25, IL-33, thymic stromal lymphopoietin, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated upon activation, normal T cell expressed and secreted, in all cells, and inhibited histamine release in HMC-1 cells. DAA controlled mitogen-activated protein kinase phosphorylation levels and the translocation of nuclear factor-kappa light chain enhancer of activated B cells into the nucleus by inhibiting IκBα decomposition in all the cells. Furthermore, DAA increased the expression of proteins involved in skin barrier functions such as filaggrin and involucrin in HaCaT cells. These results confirmed that DAA could relieve AD by controlling immune balance and recovering skin barrier function.The medicinal plant noni ( Atopic dermatitis (AD) is a chronic skin disease that affects many people worldwide . AD geneKeratinocytes maintain physical and biochemical conditions, as well as supply moisture to the skin. Keratinocytes can cause immune responses in AD through the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways ,17. TheyNF-κB increases the expression of AD-related cytokines and chemokines such as IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) ,20. TherEosinophils mainly play defensive roles against parasites and increases in their levels are observed in the peripheral blood of patients with AD . IL-5 reMorinda citrifolia), which is widely dispersed throughout Southeast Asia, the Caribbean, and Australia, has been used as a medicinal plant [Noni  matched with the standard. Content analysis indicated that the Noni extract powder contained 10.37 ± 0.94 mg/g of DAA and 0.98 ± 0.08 mg/g of AA. F. noni extract powder contained 13.11 ± 0.25 mg/g of DAA and 1.40 ± 0.08 mg/g of AA  and asperulosidic acid (AA) were provided by Phytolab . Isocove’s modified Dulbecco’s medium (IMDM), Roswell Park Memorial Institute-1640 (RPMI-1640), and Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco BRL . Fetal bovine serum (FBS) was purchased from Gemini Bio . Dimethyl sulfoxide (DMSO), dexamethasone (DEX), house dust mite (HDM), TNF-α, interferon gamma (IFN-γ), PMA, A23187, and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Inc. . A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo, Inc. . IL-1β, IL-4, IL-5, IL-6, IL-8, IL-25, IL-33, TSLP, TNF-α, and MCP-1 were obtained from R&D Systems, Inc. . TARC, MDC, RANTES, and histamine were purchased from Elabscience, Inc. . The RNA-spin Total RNA extraction kit was purchased from INtRon Biotechnology . Oligo dT primers were purchased from Bionics Inc. . The cDNA synthesis kit, SYBR green, and rox dyes were purchased from Takara Korea Biomedical, Inc. . The nuclear extraction kit was purchased from Abcam Inc. . Sodium dodecyl sulfate (SDS)-sulfate-polyacrylamide gel was purchased from Bio-Rad . The Pierce™ BCA Protein Assay Kit was purchased from Thermo Fisher Scientific, Inc. . Anti-phospho-ERK, anti-JNK, -P38, anti-β-actin, anti-lamin B1, -ERK, -JNK, -P38, -IκBα, -NF-κB, -filaggrin, and -involucrin antibodies were obtained from Santa Cruz Biotechnology, Inc. . Horseradish peroxidase-conjugated secondary antibodies were obtained from GeneTex, Inc. .Morinda citrifolia (noni) fruit was collected from the NST Bio Noni Farm Co. Ltd in French Polynesia (Indonesia islands), and F.NONI was produced in the NST bio . Briefly, for the preparation of fermented noni, fresh noni harvested in Indonesia (Java Island) was used. After washing to remove bacteria and foreign substances from fresh noni, they were frozen at −27 °C. The frozen noni stored was thawed at room temperature for two days. For the preparation of fermented noni, 2% probiotic NST 1805 (Lactobacillus plantarum) was incubated at 30 °C for 24 to 72 h, heated at 90 °C for 30 min, evaporated and concentrated, dried, and powdered (A2gen). The fruit was thoroughly washed in lukewarm water to remove fungi and to retard the growth of microbiological organisms  that were sensitive to heat. The noni fruit was then cut into pieces to fit into a 2000 mL container, and 500 mL of water was added. The chunked noni fruit was left to ferment for 24 to 72 h at 30 °C. At the end of the fermentation period, 400 mL of noni fermentation stock was transferred to a 1000 mL container. The ferment stock (400 mL) was filtered using Whatman filter paper and vacuum filtration to eliminate debris and fruit particles from the stock solution. Thirty milliliters of the filtered ferment was used for qualitative phytochemical analysis, and 10 mL was used for PDA spectroscopic analysis and optical density determination. The remaining 360 mL was used for the experiments.2O-MeOH (1:1) and mixed thoroughly, and the solution was collected into a 50 mL volumetric flask for HPLC-PDA analysis. The extracts were combined, filtered, and dried in a rotary evaporator under vacuum at 50 °C. The dried extracts were re-dissolved in MeOH for HPLC analysis. An HPLC system  equipped with an LC-20AD series pumping system and an SPD-M20A photodiode array detector (PDA) was used to analyze Noni, F. noni, and the standard iridoid (DAA and AA). Separation was carried out on a symmetry column . The binary mobile phase consisted of water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The flow rate was kept constant at 1.0 mL/min for a total run time of 40 min. The mobile phase was run with a gradient program: 0–5 min, 0% B; 5–40 min, 35% B. The sample injection volume was 5 µL. The column temperature was maintained at 25 °C. Peaks of interest were monitored at 190–380 nm using a PDA detector, and the spectra were compared with that of the standard.One gram of the f. noni powder was diluted with 50 mL of H2. HMC-1 cells were grown in IMDM medium containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) in an incubator at 37 °C and 5% CO2. The EOL-1 cells were grown in RPMI-1640 medium containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) in an incubator at 37 °C and 5% CO2.The human keratinocyte cell line (HaCaT), human mast cell line (HMC-1), and human eosinophilic leukemia cell line (EOL-1) were obtained from the Korea Cell Line Bank, Inc. . HaCaT cells were grown in DMEM containing 10% FBS and penicillin (100 units/mL)/streptomycin (100 μg/mL) in an incubator at 37 °C and 5% CO6 cells/mL and incubated for 24 h. The HaCaT cells were treated with DAA and AA at concentrations of 0.01, 0.05, 0.1, and 0.2 μM for 48 h. HMC-1 cells were seeded into 24-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. The HMC-1 cells were treated with DAA and AA at concentrations of 0.01, 0.05, 0.1, and 0.2 μM for 24 h, and the cell supernatant was aspirated. After adding 1 mL of MTT (5 mg/mL) solution to each well, the plate was incubated at 37 °C for 5 h. The MTT solution was aspirated and the crystallized formazan was dissolved in DMSO. Optical density (OD) was measured at 565 nm using an enzyme-linked immunosorbent assay (ELISA) reader . EOL-1 cells were inoculated into 96-well plates at a density of 5 × 104 cells/mL and incubated for 24 h. EOL-1 cells were treated with DAA and AA at concentrations of 3, 15, 30, and 60 nM for 48 h. Next, 10 μL of CCK-8 solution was added to each well, the plate was incubated at 37 °C for 1 h, and the OD was measured at 450 nm using an ELISA reader.The viability of HaCaT and HMC-1 cells was evaluated using the MTT assay. Briefly, HaCaT cells were seeded into 24-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. Then, they were pre-treated with DAA at concentrations of 0.01, 0.05, 0.1, and 0.2 μM for 1 h, and treated with TNF-α and IFN-γ, each at a concentration of 10 ng/mL for 24 h. HMC-1 cells were seeded into 24-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. Then, they were pre-treated with DAA at concentrations of 0.01, 0.05, 0.1, and 0.2 μM for 1 h, followed by treatment with PMACI (PMA (40 nM) + A23187 (1 μM)) for 5 h. EOL-1 cells were seeded into 96-well plates at a density of 5 × 104 cells/mL and incubated for 24 h. They were then pre-treated with DAA at 3, 15, 30, and 60 nM for 1 h, followed by treatment with HDM (10 ng/mL) for 24 h. DEX was used as the positive control. HaCaT and HMC-1 cells were treated with DEX at a concentration of 0.1 μM. EOL-1 cells were used at a concentration of 2 nM. The levels of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-25, IL-33, TSLP, TNF-α, MCP-1, TARC, MDC, and RANTES in the HaCaT, HMC-1, and EOL-1 cell supernatants were determined using ELISA kits according to the manufacturer’s instructions . HaCaT cells were seeded into 24-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. The cells were pre-treated with DAA for 1 h at concentrations of 0.01, 0.05, 0.1, and 0.2 μM. Positive control cells were treated with 0.1 μM DAA. The cells were then treated with PMA (40 nM) + A23187 (1 μM) for 8 h and the cell supernatants were collected. Histamine levels in the HMC-1 cell supernatants were determined using ELISA kit according to the manufacturer’s instructions.HMC-1 cells were inoculated into 24-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. The cells were pretreated with DAA  for 1 h, and then treated with TNF-α and IFN-γ at a concentration of 10 ng/mL for 24 h. Positive control cells were treated with 0.1 μM DEX. HMC-1 cells were inoculated into 6-well plates at a cell density of 1 × 106 cells/mL and incubated for 24 h. They were then pretreated with DAA at 0.01, 0.05, 0.1, and 0.2 μM for 1 h, followed by treatment with PMACI (PMA (40 nM) + A23187 (1 μM)) for 5 h. As a positive control, the cells were treated with 0.1 μM DEX. EOL-1 cells were seeded into 6-well plates at a density of 5 × 104 cells/mL and incubated for 24 h. They were then pretreated with DAA at 3, 15, 30, and 60 nM for 1 h, followed by treatment with HDM (10 ng/mL) for 24 h. DEX was used as a positive control at a concentration of 2 nM.HaCaT cells were inoculated into 24-well plates at a density of 1 × 10g for 5 min at 4 °C. After removing the supernatant, cold PBS was used to resuspend the cell pellet, followed by centrifugation at 1000× g for 5 min at 4 °C. The supernatant was removed, and the cell pellet was used for RNA extraction using a total RNA extraction kit. The extracted RNA was then used for cDNA synthesis using a cDNA synthesis kit and oligo dT primers. The synthesized cDNA was then used for RT-qPCR in an ABI StepOnePlus™ real-time PCR system  with SYBR Premix EX Taq. The sequences of the primers used for RT-qPCR are listed in −ΔΔC t method, based on the cycle threshold (Ct) value.After removing the supernatant, HaCaT cells were washed thrice with cold phosphate-buffered saline (PBS). After washing, the RNA was extracted using a total RNA extraction kit. Briefly, HMC-1 and EOL-1 cells were centrifuged at 1000× 6 cells/mL and incubated for 24 h. They were pretreated with DAA at 0.01, 0.05, 0.1, and 0.2 μM for 1 h, and treated with TNF-α and IFN-γ each, at a concentration of 10 ng/mL for 24 h. DEX was used as a positive control at a concentration of 0.1 μM. HMC-1 cells were seeded into 6-well plates at a density of 1 × 106 cells/mL and incubated for 24 h. Then, they were pretreated with DAA at 0.01, 0.05, 0.1, and 0.2 μM for 1 h, followed by treatment with PMACI (PMA (40 nM) + A23187 (1 μM)) for 5 h. DEX was used as a positive control at a concentration of 0.1 μM. EOL-1 cells were seeded into 6-well plates at a cell density of 5 × 104 cells/mL and incubated for 24 h. They were then pretreated with DAA at 3, 15, 30, and 60 nM for 1 h, followed by treatment with HDM (10 ng/mL) for 24 h. DEX was used as a positive control at a concentration of 2 nM.HaCaT cells were seeded into 24-well plates at a density of 1 × 10g for 10 min at 4 °C, and the supernatant was collected.After removing the culture supernatant, HaCaT cells were washed thrice with cold PBS. Nuclei were separated using a nuclear extraction kit. Proteins were extracted using a cell lysis buffer. After sonication, the cell lysates were centrifuged at 3000× g for 5 min at 4 °C. After removing the culture supernatant, the cells were washed with cold PBS. After centrifugation at 1000× g for 5 min at 4 °C, the supernatant was removed, and the nuclei were separated using a nuclear extraction kit. Proteins were extracted using a cell lysis buffer. After sonication, the cell lysates were centrifuged at 3000× g for 10 min at 4 °C, and the supernatant was collected. The cellular protein concentration in the supernatant was quantified using a BCA Protein Assay Kit . Protein samples were then subjected to 12% or 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk at ~20–25 °C for 1 h. After washing with Tris-buffered saline containing 0.5% Tween-20 (TBST), the membranes were incubated with primary antibody (1:1000 dilution) at 4 °C. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) at room temperature for 2 h. The protein bands were visualized using the ChemiDoc™ XRS system . The expression levels of the target proteins were analyzed using Image Lab statistical software (Bio-Rad) and normalized to those of β-actin and lamin B1.HMC-1 and EOL-1 cells were centrifuged at 1000× p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the normal; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.Data are shown as the mean ± standard deviation (SD). All statistical analyses were performed using one-way analysis of variance and Tukey’s honestly significant difference test. Statistically significant differences were evaluated using SPSS software . # DAA demonstrated anti-atopic activity by inhibiting gene expression and secretion of AD-related cytokines and chemokines in HaCaT, HMC-1, and EOL-1 cells. DAA regulated the expression of AD-related cytokines and chemokines by controlling MAPK and NF-kB pathways in HaCaT, HMC-1, and EOL-1 cells. In addition, DAA increased the expression of skin barrier proteins FLG and IVL in HaCaT cells. Taken together, this study suggests that DAA, the main functional ingredient in fermented noni, may be a therapeutic agent that can alleviate AD by regulating immune balance and restoring skin barrier function.


Plodia interpunctella, a severe stored-grain pest worldwide. Among the GSTs, only PiGSTd1 is antennae-specific according to both Fragments Per Kilobase Million (FPKM) and quantitative real-time PCR (qRT-PCR) analysis. Sequence analysis revealed that PiGSTd1 has a similar identity as many delta GSTs from other moths. Enzyme kinetic assays using 1-chloro-2,4-dinitrobenzene (CDNB) as substrates showed that the recombinant PiGSTd1 gave a Km of 0.2292 ± 0.01805 mM and a Vmax of 14.02 ± 0.2545 μmol·mg−1·min−1 under the optimal catalytic conditions (35°C and pH = 7.5). Further analysis revealed that the recombinant PiGSTd1 could efficiently degrade the sex pheromone component Z9-12:Ac (75.63 ± 5.52%), as well as aldehyde volatiles, including hexanal (89.10 ± 2.21%), heptanal (63.19 ± 5.36%), (E)-2-octenal (73.58 ± 3.92%), (E)-2-nonenal (75.81 ± 1.90%), and (E)-2-decenal (61.13 ± 5.24%). Taken together, our findings suggest that PiGSTd1 may play essential roles in degrading and inactivating a variety of odorants, especially sex pheromones and host volatiles of P. interpunctella.Insect glutathione-S-transferases (GSTs) play essential roles in metabolizing endogenous and exogenous compounds. GSTs that are uniquely expressed in antennae are assumed to function as scavengers of pheromones and host volatiles in the odorant detection system. Based on this assumption, antennae-specific GSTs have been identified and functionally characterized in increasing number of insect species. In the present study, 17 putative GSTs were identified from the antennal transcriptomic dataset of the Indian meal moth,  The sequence lengths, molecular weights (MWs), and isoelectric points (pI) were predicted by using ExPASy tools (https://web.expasy.org/compute_pi/)   . After sequence alignments, the phylogenetic tree was constructed by MEGA5.0 software using the neighbor-joining method with the following parameters: Poisson model, pairwise deletion, and 1,000 bootstrap replications . The homologous GST sequences were used to reconstruct a phylogenetic tree from eight species, including Plutella xylostella .Deduced amino acid sequences of GST genes from different insects were aligned with the GST sequence identified from the antennae of https://swissmodel.expasy.org/interactive). The models of PiGSTd1 were built based on the target-template alignment using ProMod3 . The secondary structure was predicted with PSIPRED software . The paired primers were forward 5′-ATGCCGGCTCAAGCCATCAA-3′ and reverse 5′-CTAATCTTTCTTCAGAAATGATGC-3′. The amplification was carried out under the conditions of denaturation at 95°C for 1 min followed by 35 cycles of 95°C for 20 s, 55°C for 20 s, and 72 °C for 1 min, and a final extension at 72°C for 5 min. The PCR products were ligated into a pEASY-Blunt E1 Expression vector  and transformed into Escherichia coli Trans-T1 , and the positive clones were isolated for expression. Cultures were started from single colonies, in LB broth with 50 μg/mL ampicillin in a 37°C shaker (220 rpm). When OD of 600 nm reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to 1 mM. After cultured for 6 h at 25°C and 220 rpm, cells were harvested by centrifugation at 8,000 g at 4°C and suspended in 20 ml of PBS buffer (pH = 7.0).PiGSTd1 expression and purification were conducted as previously described with a slight modification , which had been equilibrated with 20 mM Tris–HCl (pH = 7.9) supplemented with 100 mM NaCl, and eluted with imidazole  in an ascending series. The recombinant PiGSTd1 purity was assayed by SDS–PAGE. Its concentration was determined using Bradford's method with BCA Protein Assay Kits . Proteins were stored at −20°C before use.Km and Vmax were calculated by the linear regression of a double reciprocal plot  method , increased to 120°C at 5°C·min−1 (hold 2 min), and subsequently increased to 230°C at 10°C·min−1 (hold 5 min). The ionization current and ionization voltage were 100 μA and 70 eV, respectively. All assays were repeated three times with the heat-inactivated PiGSTd1 as the negative control. Degradation data were analyzed by one-way ANOVA (SPSS 19.0 for Windows) with Tukey's test. The least significant difference was set at P < 0.05.A GC-MS  with a DB-WAX column  was used to evaluate the degradation activities of PiGSTd1 on the main sex pheromone and environmental volatiles . The degP. interpunctella, we identified a total of 17 sequences encoding putative GSTs, which were designated as PiGSTd1-PiGSTm3. Sequence characteristics  and Blastx results are listed in X search of the best hits showed that all PiGST sequences shared relatively high sequence identities (62−97%) with their respective orthologs from other lepidopteran species , typical species in varying families, as well as congeneric Pyraloid moths. Although these GSTs were derived from diverse species, they showed relative conservation in classification. According to their sequence similarities, 17 PiGSTs were distributed into eight branches of the phylogenetic tree: delta (PiGSTd1 to PiGSTd3), epsilon (PiGSTe1 and PiGSTe2), omega (PiGSTo2 to PiGSTo4), sigma (PiGSTs1 and PiGSTs2), theta (PiGSTt1), zeta (PiGSTz1), and unclassified class (PiGSTm1)  . PiGSTd1The analyses of conserved domains among the PiGSTs revealed two domains of the protein sequences: a fairly conserved N-terminal domain and a more variable C-terminal domain among different subclasses . BesidesBased on qRT-PCR determination, only PiGSTd1 expression was antennae-specific, and its expression level was significantly higher in males than in females , indicatN-terminal domain, a C-terminal domain, and a linker in between  of thioredoxin fold-served as the glutathione binding site (G-site). Ser40 in PiGSTd1 appeared to be responsible for enzyme catalysis.According to a multiple alignment of PiGSTd1 with delta GSTs from other moths, PiGSTd1 showed relatively high identities (63.11–68.83%) with HvGSTd1 (AWX68884.1), OfGSTd1 (QIC35737.1), CsGSTd1 (AKS40338.1), SeGSTd1 (ASN63930.1), BmGSTd1 (NP_001037183.1), and PrGSTd1 (APW77568.1) , indicat between . In the E. coli strain BL21 through pEASY-Blunt E1 vector. SDS–PAGE showed that Ni+-column-purified PiGSTd1 displayed a single band with a MW of ~27kDa -2-octenal (73.58 ± 3.92%), (E)-2-nonenal (75.81 ± 1.90%), and (E)-2-decenal (61.13 ± 5.24%). The results indicated that PiGSTd1 highly expressed in P. interpunctella antennae was involved in degrading sex pheromones and host volatiles.The ability of recombinant PiGSTd1 to degrade odorants was evaluated by GC-MS. The results showed that PiGSTd1 more efficiently degraded the sex pheromone component Z9-12:Ac (75.63 ± 5.52%) as compared with the pheromone analog Z8-12:Ac (58.47 ± 1.64%), despite only a differently positioned double bond. Besides sex pheromones, PiGSTd1 also displayed high efficiency in degrading various host odorants and environmental volatiles , e.g., αP. interpunctella, which is more than the number identified from the antennae of C. suppressalis (16 genes)    -8-dodecenyl alcohol in antennae of Grapholita molesta (75.01%) -3-hexenol -2-octenal (73.58 ± 3.92%), and (E)-2-nonenal (75.81 ± 1.90%), which could attract P. interpunctella  and flower fragrance , with degradation rates of 30.91 ± 5.17% and 13.97 ± 2.76%, respectively. Presumably, P. interpunctella infests processed foods and inhabits indoor areas, resulting in low degradation against green leaf volatiles and flower fragrances. However, how PiGSTd1 affects the olfactory recognition of P. interpunctella remains to be investigated in vivo.Besides degrading sex pheromones, insect delta GSTs also play roles in degrading host volatiles and environmental odorants  can be found in the article/The research was designed by JY and CL. The experiments were performed by HL, YT and HS. Data were analyzed by YT and QW. HL and YT wrote the manuscript. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To the Editor:,The ideal extracorporeal circuit for kidney replacement therapy (CKRT) would have no systemic anticoagulation. However, this approach runs the risk of extracorporeal circuit loss or partial loss of dialyzer fibers. Regional citrate anticoagulation provides anticoagulation of the circuit owing to citrate chelating calciumRecent pandemic conditions have posed additional challenges to the use of CKRT in intensive care units. Hypercoagulability, filter loss, nursing workload, and personal protective equipment needs associated with CKRT have all rendered intermittent therapy increasingly preferable. HD is widely considered to be comparable to CKRT.An ex vivo dialysis circuit was set up using donated whole human blood. Ethics committee approval was not required because we used expired donated blood. Dialysis was performed using a Fresenius 5008 machine  with online-generated ultrapure dialysate in hemodiafiltration post-dilution mode. A conventional and affordable high-flux synthetic polymer dialyzer  was used. Dialysate flow was 500 mL/min, blood pump speed 150 mL/min, and substitutive rate 25 mL/min. Calcium dialysate concentration was increased in increments of 0.25 mmol/L using a standard hemodialysis additive (Baxter Corporation). Anticoagulation was obtained using citrate dextrose solution. Citrate was infused into the heparin infusion port at 200 mL/h and ultrafiltration rate was set at 180 mL/h to account for citrate infusion. Blood samples were obtained pre-dialyzer (n = 4) and post-dialyzer (n = 16) and were analyzed using an i-STAT 1 Analyzer (Abbott Point of Care Inc.). We assessed the following: (1) ionized calcium stability with a fixed calcium dialysate concentration of 1.5 mmol/L. Ionized calcium was tested every hour at 1.5, 2.5, and 3.5 hours. (2) Ionized calcium changes after every 0.25 mmol/L change of calcium dialysate concentration with initial calcium dialysate concentration from 1.25 mmol/L to 2.0 mmol/L (n = 4). Samples were collected every hour (n = 4) and/or 10 minutes after every calcium dialysate concentration change (n = 8).P = 0.001 (The mean ± standard deviation (SD) pre-dialyzer ionized calcium was 0.26 ± 0.02 mmol/L. The mean ± SD post-dialyzer ionized calcium was 1.01 ± 0.09, 1.22 ± 0.07, 1.39 ± 0.07, and 1.55 ± 0.15 mmol/L for calcium dialysate concentration at 1.25, 1.5, 1.75, and 2 mmol/L, respectively. Ionized calcium was tightly correlated to dialysate calcium concentration, r = 0.99,  = 0.001 A. No cloP = 0.98. For a fixed calcium dialysate concentration of 1.5 mmol/L, ionized calcium was 1.28 mmol/L, 1.25 mmol/L, and 1.23 mmol/L, after 1.5, 2.5, and 3.5 hours, respectively  compared to every 10 minutes , ectively B.These experiments demonstrate that ionized calcium levels can be managed by modulating calcium dialysate concentrations without the need to administer additional calcium intravenously. We report stable ionized calcium over time to ensure a pre-dialyzer ionized calcium <0.3 mmol/L. We also demonstrated that calcium dialysate concentration changes lead to an immediate modification of ionized calcium levels, since these values were achieved after only 10 minutes, with mass transfer of calcium from dialysate to blood self-regulating as product of the changing relative concentrations within the 2 compartments.Recent studies have shown patients infected with COVID-19 are severely hypercoagulable,We present an alternative option to maintain a calcium level target by modulating calcium dialysate concentration, in combination with limited replacement derived from the administration of online-generated dialysate in hemodiafiltration mode, without the need of additional venous calcium infusion. Such an approach is globally available, and is feasible from both a financial and workload perspective. This new protocol could be easily implemented in all HD patients at high risk of bleeding or with heparin contraindications.


Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD ( N–glycosidically linked oligosaccharides [SERPINA1 gene [Alpha-1 antitrypsin (AAT) is an abundant plasma protein that plays a key role in the innate immune response. The majority of circulating AAT is produced by hepatocytes and is generated as a single polypeptide chain that is post-translationally modified by glycosylation through the addition of charides . AAT defNA1 gene . The mosNA1 gene ,5,6. TheNA1 gene . The codNA1 gene . MoreoveNA1 gene . FrequenNA1 gene , leadingNA1 gene .4 [The primary role of the AAT protein is to function as a serine protease inhibitor, as AAT has been shown to inhibit a range of proteases derived from degranulating neutrophils, including neutrophil elastase (NE) . New ins4 , tumor n4 , C-X-C m4 , interfe4  and inte4 . The ant4 . Recently we established that a significant proportion of glycosylated AAT is bound to other abundant plasma proteins in the circulation. We identified an interaction between AAT and complement component C3 and demonstrated that a by-product of C3 cleavage, C3d, is present at increased levels in the plasma of AATD individuals . Our finAll chemicals and reagents were of the highest purity available and were purchased from Sigma-Aldrich, Dublin, Ireland unless specified otherwise.® kit on the Sebia Hydrasys® System. HC (AAT sufficient) individuals (n = 40) were defined as having an MM phenotype with plasma AAT levels within the normal range (1.1–1.8 g/L) (n = 88) were recruited from the National AATD clinic in Beaumont Hospital and were confirmed as having a ZZ or MZ phenotype. AATD individuals were stable with no exacerbations at the time of recruitment. AAT sufficient COPD patients (n = 10) were recruited from the general respiratory clinic in Beaumont Hospital and confirmed as having an MM phenotype with plasma AAT levels within the normal range  and written informed consent was obtained from all study participants. The inclusion criteria for patient participants included: (1) AATD confirmed by isoelectric focusing; (2) COPD with MM-AAT phenotype; (3) aged ≥18 years old, and (4) patients capable of giving informed consent. For healthy controls (HC) the inclusion criteria included: (1) aged ≥18 years; (2) normal AAT-MM phenotype and AAT level, and (3), no respiratory illness. The exclusion criteria for all participants included: (1) aged <18 years; (2) unable to give consent; (3) pregnant or breast-feeding women, and (4), active tobacco smoking. AAT phenotyping was carried out by isoelectric focusing using the Hydragel 18 A1AT isoelectricfocusing1.8 g/L) . All weral range . ® tubes containing lithium heparin and centrifuged at 350× g for 5 min at room temperature. Plasma was aliquoted for immediate use or directly stored at −80 °C. Neutrophils were isolated from whole blood samples by dextran sedimentation and Lymphoprep  centrifugation as previously described [Blood samples were collected in Sarstedt-Monovetteescribed . Purifieescribed ,29. Neutw/v) NuPAGE gels. After electrophoresis, gels were stained by Coomassie Blue R250 or were silver-stained for visualization of proteins, or alternatively, proteins were transferred onto 0.2 μm nitrocellulose or PVDF membrane by Western blotting with a semidry blotter for 1 h at 100 mA. The efficient transfer was verified by Ponceau S staining of membranes followed by blocking of membranes in PBS-Tween (0.05% v/v) containing 3% (w/v) nonfat dried milk and 1% (w/v) bovine serum albumin (BSA). SDS–PAGE analyses of samples were carried out under denaturing or non-reducing conditions. Samples were electrophoresed with 12.5% ; mouse monoclonal BPI ; goat polyclonal MMP-9 ; rabbit polyclonal hCAP-18 , or mouse monoclonal β-actin . Relevant secondary antibodies were all horseradish peroxidase (HRP) linked anti-goat, anti-rabbit or anti-mouse . Immunoreactive bands were visualized with chemiluminescent HRP substrate (Millipore). Images and densitometry were obtained on the Syngene G: BOX Chemi XL gel documentation system.v/v) HC or ZZ-AATD fresh plasma and incubated for 1 h at 37 °C. Samples were analyzed on 12.5% (w/v) SDS-PAGE gels under non-reducing conditions, and gels were either silver stained or Western blotted for C3d using a mouse anti-C3d antibody . Purified C3d was included as a positive control . Production of C3d by NE cleavage of total C3 was evaluated by adding 338 nM NE  to 1% (6/mL) were fixed in 4% (w/v) paraformaldehyde for 10 min. Cells were then washed in PBS and blocked for 30 min with 1% (w/v) BSA in the presence of protease inhibitors to maintain plasma membrane receptor integrity (20 µg/mL phenylmethane sulfonylfluoride (PMSF), 10 µg/mL Na-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), 10 µg/mL pepstatin A, 200 µg/mL Pefabloc, and 10 µg/mL leupeptinhemisulfate). After 1 h of incubation with primary antibodies, samples were washed and incubated for 30 min with relevant secondary FITC-labeled antibodies. Primary antibodies included 1 μg/mL mouse monoclonal anti-CD11b (CR3)  or mouse monoclonal anti–C3d . FITC-labeled mouse IgG kappa binding protein (m-IgGκ BP) (Santa Cruz Biotechnology) or FITC goat anti-mouse IgG (Santa Cruz Biotechnology) were used as secondary FITC-labeled antibodies (Santa Cruz Biotechnology).Levels of neutrophil plasma membrane C3d and cognate receptor CR3 were evaluated by flow cytometry. For this, isolated neutrophils  (1 mM), for 30 min ,30. Subs7 cells/mL), cells isolated from HC individuals were challenged with increasing concentrations of C3d (0–40 µg/mL) for 10 min at 37 °C. Extracellular supernatants were then analyzed for degranulated protein as previously described [7) was included as a positive control as it has previously been reported to cause secondary and tertiary granule release, but not primary granule degranulation [To assess degranulation of tertiary, secondary, and primary granules of circulating neutrophils . An NE-specific FRET substrate was used  [Ex vivo analysis of MPO (Cambridge Bioscience), lactoferrin (Cambridge Bioscience), and BPI  was measured in plasma using relevant quantitative sandwich enzyme-linked immunosorbent assays (ELISA), as per the manufacturer’s instructions.v/v) heat-inactivated fetal calf serum (FCS)  and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2. Cells (2.5 × 106) were added to a 12 well 3.8 cm2 round culture dish for treatment after being washed and resuspended in RPMI without FCS. TNF-α (0.5 ng) was employed as a positive control in a dose-response experiment with increasing concentrations of C3d protein  (0–20 µg/mL). Following 6 h treatment, cells were centrifuged at 500× g for 5 min to obtain cell-free supernatants, and a CXCL8 ELISA (Cambridge Bioscience) was performed. A standard curve was created in accordance with suppliers’ instructions and results recorded at 405 nm on a Spectra Max M3 .The HL-60 neutrophil-like cell line  was cultured in complete RPMI media containing 10%  (Sigma) were grown in Endothelial Cell Growth Medium in a humidified incubator at 37 °C with 5% COette tip . Cells wt-test was used where distribution was normal and when comparison was being made between two groups. The Mann–Whitney U test was employed where data were not normally distributed. Two-way ANOVA was used for independent group comparisons, followed post-hoc by Bonferroni’s multiple comparison test where appropriate. Statistical significance was determined with a p-value < 0.05. The data were analyzed with Graphpad Prism 8.0 and SPSS software.Results were expressed as mean ± SEM of biological replicates or independent experiments as stated in the figure legends. Data were tested for normality using the D’Agostino and Pearson omnibus normality test. A Student’s n = 20), MZ-AATD (n = 8), MM-COPD (n = 10), and MM-HC individuals (n = 17) by ELISA. The mean C3d concentration was significantly increased in ZZ-AATD (~2 µg/mL) compared to MM-HC (p < 0.0001), confirming that complement activation and C3d production are increased in ZZ-AATD individuals (p = 0.0002) and AAT-sufficient MM-COPD patients compared to ZZ-AATD (p < 0.0001), indicating that the increase in complement activation is related to a lack of AAT, as previously reported [To confirm complement activation in AATD, quantification of C3d was performed in plasma of ZZ-AATD  (p = 0.636), further supporting the concept that the increase in C3d in ZZ-AATD was due to a lack of AAT rather than inflammatory lung disease. Furthermore, FEV1 (% predicted) values were obtained for each patient from the Irish National AATD Registry and correlated with neutrophil membrane-bound C3d . This suggests complement activation as a key player in the pathogenesis of AATD and is in line with the absence of an adequate anti-protease protective screen. Plasma and neutrophils of AATD patients homozygous for the Z-allele were utilized in all subsequent analyses. As neutrophilic inflammation is a prominent feature of AATD, ensuing experiments assessed an association between raised plasma C3d levels and circulating neutrophils. By flow cytometry, C3d was found significantly increased on the outer membrane surface of circulating neutrophils of ZZ-AATD compared to MM-HC individuals (= 0.038) B. Moreovound C3d C. A signv/v) HC or AATD plasma, and reactions were incubated for 1 h at 37 °C. Protein samples were then separated under non-reducing SDS-PAGE conditions, and the gels were silver-stained (p = 0.001)  B. This r6) were stimulated with PMA (1 µg/mL) at 37 °C for 10 min. Membrane-bound NE was assessed by FRET analysis, with significantly increased levels of active NE detected on neutrophil outer plasma membranes following PMA challenge (p = 0.01) (p = 0.002)  C. Moreov= 0.002) D. p = 0.011 and p = 0.0001, respectively)  C. Collec7) isolated from HC individuals were challenged with increasing concentrations of C3d (0–40 µg/mL) or TNF-α (5 ng/1 × 107) for 10 min at 37 °C. TNF-α was used as a positive control as it has previously been reported to cause secondary and tertiary granule release from neutrophils [p = 0.018), 20 (p = 0.037) or 40 µg (p = 0.042) C3d (p = 0.004) (p = 0.013), or 40 µg C3d (p = 0.025)  C3d A,B. At t= 0.004) A,C. Of n= 0.025) A,D.p < 0.001) (p = 0.02) (p < 0.0001)  A. This s = 0.02) B. Finall 0.0001) C. Collec6) as a positive control for CXCL8 release. Following 6 h treatment, extracellular culture supernatants were collected and analyzed for CXCL8 by ELISA. HL-60 cells released significantly more CXCL8 in response to 5 µg  and 20 µg C3d  compared to untreated cells (413.4 ± 35 pg/mL)   A. In cor 0.0001) B. These in vitro wound-healing assay was used to examine the effect of C3d on endothelial cell migration, a key process in wound healing and angiogenesis. HUVEC cells were grown to confluence in 24-well plates, and a single scratch was created down the center of the well to produce a wound. Then, cells were incubated with or without C3d treatment, with images recorded at 0, 4, and 8 h to assess wound closure. For the current set of experiments, we employed 10 or 20 μg/mL C3d, which was within the physiological range. HUVEC untreated controls showed more evidence of cell scatter-driven migration compared to cells in the presence of C3d (20 µg) after 8 h incubation (p < 0.01) (p < 0.0001), or those treated with C3d (20 µg) only (p < 0.0001)  A,B. More 0.0001) C,D. CollNeutrophilic inflammation is a prominent feature of both COPD and AATD. However, neutrophils in AATD are a clinically important cell to study as patients have a significantly higher burden of neutrophils in their airways ,40 and ain vitro and in vivo AATD models would help determine whether C3d elevation is a driver or an effect. Moreover, these results demonstrate that the in vitro challenge of neutrophils with C3d is a potent stimulus resulting in the release of all three granule types, which is in contrast to TNF-α, which is effective at secondary and tertiary release only. Additionally, further experiments confirming the ability of C3d to cause CXCL8 release could compare the impact to C3a, an additional inflammatory marker related to complement activation [Dysregulated neutrophil degranulation is an important feature in the pathogenesis of AATD. It has previously been demonstrated that AAT can modulate TNF-α induced degranulation processes in a dose-dependent manner, with increased plasma levels of secondary and tertiary neutrophil granule components detected in AATD . Moreovetivation . C3a is tivation .The recorded induction of granule release by C3d may play a role in the pathogenesis of AATD, including degradation of tissue by excessive release of proteases or aiding in the development of autoantibodies. For example, the tertiary granule protein MMP-9 can degrade major extracellular matrix structures, including laminin and collagen , and is in vitro data demonstrate that endothelial cell migration was significantly reduced in the presence of C3d and neutrophil granule proteins. Collectively, these data indicate that targeting CR3/C3d signaling in neutrophils, thereby modulating the C3d cycle of inflammation, may represent a new therapeutic avenue in AATD-related disease.Complement activation has been reported to play a role in ANCA-associated vasculitis, with circulating levels of complement activation fragments increased in the plasma of those with active disease . Of releIncreased complement activation and C3d levels have been demonstrated to represent key processes in the pathogenesis of AATD. Greater C3d concentrations retain the ability to bind CR3 on the neutrophil membrane resulting in increased release of primary, secondary, and tertiary granule components. C3d also induces the release of CXCL8, a key neutrophil chemoattractant, potentially amplifying the neutrophilic inflammatory burden. Moreover, within the blood vasculature, the presence of C3d and granule proteins may slow endothelial cell migration and wound repair. As AAT augmentation therapy impacts C3d production , this st


P < 0.05) in fat, fat free dry matter (FFDM), and moisture contents across the treatment. Fresh beef sausage fortified with 70% EMW had the highest fat contents (25.7 ± 0.83%) while those fortified with 10% fat (T3) had the highest FFDM (55.85 ± 0.57%) and moisture content (69.15 ± 0.62) compared to other treatments. In addition, among individual saturated fatty acids, beef sausage fortified with 50% meat wastes (T1) revealed significantly higher palmitic acid (31.06 ± 0.13), stearic acid (22.52 ± 0.29), myristic acid (3.84 ± 0.05), and lauric acid (0.04 ± 0.05) and the lowest margaric (0.98 ± 0.02) contents as compared to treatments T2 and T3. Also, beef sausage containing 10% fat showed the lowest (P < 0.05) saturated fatty acid (SFA) and higher monounsaturated (MUFA), polyunsaturated fatty acid (PUFA), n-6, n-3, PUFA : SFA, PUFA/MUFA, n-6/n-3, and desaturase indexes (DI) compared to treatments T1 and T2. Frying with sunflower oil significantly increased PUFA, n-6, n-6/n-3, and desaturase indexes and lowered SFA, n-3, and PUFA/SFA compared to frying with olive oil. In relation to raw beef sausage, frying with oil substantially increased the amount of MUFA, PUFA, n-6, and PUFA/SFA but reduced SFA content across the treatments.Application of cooking oil during thermal processing can influence the nutritional qualities of meat products during consumption. This study determined the effect of frying with sunflower and olive oil on the fatty acid profile of sausage fortified with edible meat waste (EMW) as a fat replacer was evaluated. Fresh beef sausages were formulated in ratios of 30% lean meat (LM) and 70% EMW, 50% LM and 50% EMW, and 90% LM and 10% fat (control) and designated as T1, T2, and T3, respectively. The proximate analysis results revealed significant differences ( Meat is one of the most important sources of nutrients  widely consumed by humans in both developed and developing countries across the world. The preference for meat and meat product consumption could be largely attributed to its taste, flavour, juiciness, palatability, and ability to provide high biological values in human diet –4. Over Also, Estévez et al.  and RamiResearch studies indicated that vegetable oil represents a well-known dietary source of energy. The utilization of vegetable oil in meat products is effective in lowering the amount of saturated fatty acid contents thereby giving a positive effect on human health , 15. StuPresently, the interest of many researchers is to produce meat products, such as meat sausage, that can promote human health conditions. Meat sausages are usually made from ground meat  along with salt, spices, and other flavouring ingredients. In addition, products such as pork back fat, camel hump fat, beef fat, edible animal by-products, and vegetable oils have been incorporated into meat sausages with specific amount to enrich their textural and sensory quality and nutritional content and reduce their production cost , 19. TheThe study was conducted in a commercial abattoir in East London situated under Buffalo City Metropolitan Municipality and Meat Science Laboratory at the University of Fort Hare. The East London abattoir is located 120 km away from Alice where the University of Fort Hare is established at latitudes and longitudes of 32.97°S and 27.87°E, respectively. The East London abattoir is a high throughput equipped with sophisticated machines and equipment fit for a standard abattoir.The lean beef meat and edible meat waste (EMW) were collected separately from slaughtered cattle at the abattoir to produce a novel beef sausage. Samples were packed in low-density polyethylene plastic bags and kept in a cooler and transported to the laboratory within 180 minutes. The EMW refers to meat waste generated during the cutting and trimming of the meat at the abattoir, and this usually consists of tendons, connective tissues, and trimmed meat fat. In the production of sausage, the lean beef and edible meat waste samples were combined in ratios of 30 : 70 and 50 : 50 and designated as T1 and T2, respectively, while the control contained 90% lean meat and 10% fat as treatment 3 (T3). Samples from each treatment were first chopped into 0.00129 meter square, mixed with seasonings (spices (0.33%), salt (0.19%), and ice (0.33%)), and then minced through a 5 mm plate using a mincer . Sausage batter was then pumped and stuffed lightly into a 25 mm diameter sheep sausage casing  with Tre Spade sausage filler tools to produce average weight of 125 g each.Fresh sausage from each treatment was then fried at 160-180°C for 5-10 minutes using two types of cooking oils (olive or sunflower oil). Doneness was determined by inserting a probe thermometer (ThermoPro TP food thermometer) into a geometrical centre of the sausage to measure its internal temperature during frying. The samples were considered done when the digital thermometer gave an alarm and flashed green light. The samples were drained, cooled at room temperature, vacuumed packed, and stored at -80°C until fatty acid analysis was carried out. All sausage samples were analysed in triplicate per treatment and per cooking oil type for fatty acid analysis.w/w) per 100 g tissue. Thereafter, the extracted fat muscle was stored in a polytop  under a blanket of nitrogen and frozen at –20°C for analysis of fatty acids.Total lipid of the raw and cooked sausage samples was quantitatively extracted as described by Folch et al. , using cμm film thicknesses). Analysis was performed using an initial isothermic period (40°C for 2 minutes). Thereafter, temperature was increased at a rate of 4°C/minute to 230°C. Finally, an isothermic period of 230°C for 10 minutes was followed. FAME n-hexane (1 μl) was injected into the column using a Varian CP 8400 Autosampler. The injection port and detector were both maintained at 250°C. Hydrogen, at 45 psi, functioned as the carrier gas, while nitrogen was employed as the makeup gas. Galaxie Chromatography Data System Software recorded the chromatograms.A lipid aliquot (±30 mg) of sausage batter lipid was converted to methyl esters by base-catalyzed transesterification, in order to avoid CLA isomerization, with sodium methoxide (0.5 M solution in anhydrous methanol) during 2 h at 30°C, as proposed by Park et al. , Kramer Fatty acid methyl ester samples were identified by comparing the retention times of FAME peaks from samples with those of standards obtained from Supelco . Conjugated linoleic acid (CLA) standards were obtained from Matreya Inc. . These standards included cis-9 and trans-11 and trans-10 and cis-12-18:2 isomers. Heneicosenoic acid (C21:0) was used as the internal standard to improve quantitative FAME estimation.9 desaturase index (C18:1c9/C18:0), total omega-6, total omega-3, and the ratio of omega-6 to omega-3 (n-6)/(n-3) FAs. The atherogenicity index (AI) was calculated as AI = (C12 : 0 + 4 × C14 : 0 + C16 : 0)/(MUFA + PUFA) [Fatty acids were expressed as the proportion of each individual fatty acid to the total of all the fatty acids present in the sample. Fatty acid data were used to calculate the following ratios of FAs: total SFAs, total MUFAs, total PUFAs, PUFA/SFA, ∆ + PUFA) .P < 0.05.The Statistical Analysis System (SAS version 9.1.3 of 2007) was used for data analysis. The PROC GLM procedure of SAS was used to consider the effect of the types of cooking oil (olive oil and sunflower oil) and meat type  on the fatty acid beef sausage. Significant differences between the least square means for types of cooking oil were measured using Fishers' least significance difference (LSD) method of SAS, with a significance level of P < 0.05) in intramuscular fat, fat free dry matter (FFDM), and moisture contents across the treatment. As expected, beef sausage containing 70% edible meat wastes (T1) had higher fat content (25.7 ± 0.83%) than other treatments. Beef sausage fortified with 10% fat (T3) had the lowest fat content but highest FFDM (55.85 ± 0.57%) and moisture content (69.15 ± 0.62) than other treatments. A similar trend was also observed in the fried beef sausage (P < 0.05) higher fat content (18.36%) and lower FFDM (25.93%) and moisture (55.91%) contents compared to those cooked with olive oil  in individual fatty acid composition of raw beef sausage across the treatments. Among individual saturated fatty acids, beef sausage fortified with 50% meat wastes (T1) revealed significantly higher palmitic acid (31.06 ± 0.13), stearic acid (22.52 ± 0.29), myristic acid (3.84 ± 0.05), and lauric acid (0.04 ± 0.05) and the lowest margaric (0.98 ± 0.02) contents compared to treatments T2 and T3. On the other hand, the individual monounsaturated fatty acid showed that beef sausage fortified with 10% fat (T3) had higher oleic (39.25 ± 0.34), vaccenic (0.52 ± 0.02), and heptadecenoic (0.67 ± 0.02) content and the lowest myristoleic (0.45 ± 0.01) and palmitoleic (2.50 ± 0.05) content as compared to treatments T1 and T2. The individual polyunsaturated fatty acid content was significantly higher in beef sausage containing 10% meat wastes (T3) as compared to treatments T1 and T2. In general, it was observed that the values of total monounsaturated fatty acid, polyunsaturated fatty acid, n-6, n-3, PUFA : SFA, PUFA/MUFA, n-6/n-3, and desaturase indexes (DI) were higher in beef sausage fortified with 10% fat than in other treatments. Meanwhile, the beef sausage fortified with 50% edible meat wastes (T2) had higher saturated fatty content and atherogenicity index (AI) compared to treatments T1 and T3. Thus, the findings in this study were in agreement with the Food and Agriculture Organization of the United Nations and World Health Organization, where the values of the n-3 : n-6 ratio and PUFA : SFA for raw beef sausages fell within the recommended ratio of ≥1 and >0.04, respectively, as required in human diet [man diet –31. HoweP < 0.05). As expected, beef sausage containing 10% fat (T3) had the lowest total SFA and highest MUFA and PUFA compared to treatments T1 and T2. In comparison to the raw beef sausage, cooking of the meat samples by frying significantly decreased the percentage of individual SFA values while increasing the individual MUFA and PUFA values as well as n-6, n-3, PUFA : SFA, PUFA/MUFA, n-6/n-3, and atherogenicity (AI) and desaturase index values across the treatments. These results are similar to the findings of Cunningham et al. [P < 0.05). On the other hand, cooking of beef sausage with sunflower oil revealed higher PUFA, n-6, n-3, PUFA : SFA, PUFA/MUFA, n-6/n-3, and atherogenicity (AI) and desaturase index values than cooking with olive oil. These differences could be attributed to the individual fatty acid content of the cooking oil. A report has shown that the type of vegetable oil used during cooking can significantly influence changes in the fatty acid composition of meat products [α-linolenic acid concentrations compared to those eating diet cooked with sunflower oil [As shown in m et al.  who repoproducts . Sunflowproducts , while oproducts . Other sproducts . Similarproducts  also fouower oil .Despite the increase in the omega-3 : omega-6 fatty acid ratio in the sausage treatments after frying with the oils, the mean value of the omega-3 : omega-6 fatty acid ratio was greater than 1 : 5 and this was within the FAO/WHO recommended range. Furthermore, the significant reduction in saturated fatty acids after cooking showed that the sausage could have a positive influence on the human health when consumed. Therefore, it may be concluded that using sausage made with edible meat waste as a fat replacer may not have any negative effects on human if consumed.