Dehalobacterium and Dehalogenimonas, and nZVI as a treatment train to detoxify chlorinated methanes , and 1,2-DCA. First, we showed that CF (500 μM) was fully degraded by 1 g/L nZVI to DCM as a major by-product, which was susceptible to fermentation by Dehalobacterium to innocuous products. Our results indicate that soluble compounds released by nZVI might cause an inhibitory impact on Dehalobacterium activity, avoiding DCM depletion. The DCM dechlorination activity was recovered when transferred to a fresh medium without nZVI. The increase in H2 production and pH was discarded as potential inhibitors. Similarly, a Dehalogenimonas-containing culture was unable to dichloroeliminate 1,2-DCA when exposed to 1 g/L nZVI, but dechlorinating activity was also recovered when transferred to nZVI-free media. The recovery of the dechlorinating activity of Dehalobacterium and Dehalogenimonas suggests that combination of nZVI and bioremediation techniques can be feasible under field conditions where dilution processes can alleviate the impact of the potential inhibitory soluble compounds.Nanoscale zero-valent iron (nZVI) has the potential to degrade a diversity of chlorinated compounds, and it is widely used for remediation of contaminated groundwaters. However, some frequently detected contaminants such as dichloromethane (DCM) and 1,2-dichloroethane  have shown nearly no reactivity with nZVI. Here, we tested the feasibility of combining anaerobic dechlorinating bacteria, The online version contains supplementary material available at 10.1007/s11356-023-25376-z. At the heart of this process are membrane-bound reductive dehalogenases, which use chlorinated compounds as electron acceptors to generate cellular energy while using H2 as electron donor for organohalide respiration  aqueous solution and a 0.9 M ferrous chloride (III)  aqueous solution. Both solutions were prepared using ultrapure water (Milli-Q). Water used to prepare the FeCl3 solution was previously deoxygenated by bubbling with N2. Then, the NaBH4 solution was added dropwise to the FeCl3 solution while stirring at 200 rpm and bubbling with N2. Then, nZVI was washed three times using anaerobic ultrapure water, and it was finally stored in a serum bottle and sealed with Teflon-coated butyl rubber aluminum crimp caps until use to keep anaerobic conditions.Particles of nZVI were synthesized as described elsewhere  estuary sediments and slurry samples collected from a membrane bioreactor operating in a centralized industrial wastewater treatment plant, respectively. Both cultures were cultivated for more than 5 years in an anaerobic defined medium described elsewhere  and Na2S·9H2O and L-cysteine (0.2 mM each) as reducing agents. Dehalobacterium cultures also contained tungsten (22.8 μM) and selenium (24.2 μM). Yeast extract (200 mg/L) and acetate (5 mM) were added to the Dehalobacterium and Dehalogenimonas cultures, respectively. Microcosms containing Dehalobacterium were gassed with N2 (0.4 bar overpressure), and Dehalogenimonas cultures were gassed with N2/CO2  and H2 (added to an overpressure of 0.4 bar) unless otherwise stated. Both media were buffered with bicarbonate solution (0.01 M). When indicated, either Tris-HCl  or HEPES (60 mM) was added as buffer solutions.The cultures containing Dehalobacterium and Dehalogenimonas were inoculated by transferring 3 mL and 5 mL from cultures during the exponential degradation phase of DCM and 1,2-DCA, respectively. The microcosms containing nZVI were not gassed with H2 but with N2/CO2 . Biotic controls were cultivated under static conditions; biotic-abiotic treatments and abiotic controls were incubated under shaking conditions (150 rpm) with the aim of maintaining the nanoparticles in suspension. All treatments were maintained at 25 °C in the dark.Sixty-five mL of the anaerobic medium described above was added into 100-mL serum bottles and sealed with Teflon-coated butyl rubber septa aluminum crimp caps. When indicated, some of the medium components were omitted. At least three treatments were included for each experiment: (i) abiotic controls containing the anaerobic defined medium and nZVI, (ii) biotic controls containing the anaerobic defined medium and the corresponding bacterium, and (iii) biotic-abiotic treatments containing the anaerobic defined medium, nZVI, and the corresponding bacterium. Each microcosm was amended with either 1,2-DCA, CF or DCM . In the biotic treatments, hexamethyldisilazane.The morphological features of nZVI and the interaction between cells and nZVI particles were analyzed with a Zeiss Merlin Field-Emission Scanning Electron Microscope (SEM). Samples were prepared as follows: a drop from the sample was placed in silicon chips, where it was then fixed with 2.5% glutaraldehyde in phosphate buffer 0.1 M for 2 h at 4 °C, post-fixed with 1% osmium tetroxide with 0.8% potassium ferrocyanide for 2 h, and dehydrated in increasing concentrations of ethanol . Finally, it was chemically dried with L32 ratio, which corresponds to a defined oxidation state of iron according to Tan et al.  to measure the oxidation state of nZVI. A FEI Tecnai G2 F20 HR(S) was used. This TEM was equipped with a Gatan Image Filter (GIF) Quantum SE963 operated at 200 kV to obtain energy electron loss spectroscopy (EELS) analysis. This analysis defines an n et al. . Accordicis-1,2-dichloroethene, vinyl chloride, chloroethane, methane, ethene, and 1,2-DCA, the initial oven temperature was 35 °C; it was held for 3 min and then ramped at 10 °C/min to 240 °C, which was held for 4 min  fitted with an Agilent DB-624 column (30 m × 0.32 mm with 0.25 μm film thickness) and a flame ionization detector. Helium was used as the carrier gas (0.9 mL/min), and the injector and detector temperatures were set at 250 and 300 °C, respectively. To analyze CF, propene, We first studied the abiotic dechlorination of CF, DCM, and 1,2-DCA by nZVI and the effect of omitting vitamins (V−) and sulfur-cysteine (S−) from the complete medium (CM) described in the “nZVI synthesis and bacterial cultures” section.Based on previous reports, CF reacted with nZVI producing mainly DCM (Song and Carraway − or bicarbonate (present in our culture medium) can deactivate reaction sites in the nZVI surface , and DCM was detected as a byproduct at very low concentrations (≤ 10 μM) after 115 days Fig. , B. In mcis-1,2-dichloroethene, and 1,2-DCA  and 1,2-DCA (500 μM) was assessed using 1 g nZVI/L. Consistent with previous reports  by a Dehalobacterium was not observed with either 0.5 or 1 g nZVI/L, while biotic microcosms fully fermented DCM in 4 days , which could favor the decomposition of functional groups in proteins and lipopolysaccharides from cell membranes  compared with cultures without nZVI (4.9 ± 0.1) , 8.5, and 9 without nZVI. Degradation of DCM in cultures at pH 7 and 8.5 occurred at similar degradation rates after a lag phase of 6 days, but cultures at pH 9 exhibited an extended lag phase of 18 days before DCM was fully consumed  Fig. . To deteDehalobacterium activity, we buffered the Dehalobacterium-containing cultures with 25 mM Tris-HCl (pH = 7.5) and amended 1 g nZVI/L, but degradation of DCM was not observed for 12 days although pH was 8.2 ± 0.3 . It provides evidence that H2 accumulated because of water hydrolysis by nZVI even though hydrogenotrophs were present in the bacterial consortium  found inhibitory for DCM fermentation in Dehalobacterium , ii) particles of nZVI were removed from microcosms with no addition of H2 , and iii) particles of nZVI were not removed (control). All treatments were amended with 2000 μM DCM after CF depletion.We prepared nine abiotic-biotic microcosms containing y 7 Fig. , three d2 concentration was 4 ± 1 mM  , and complete degradation of DCM was observed after a lag phase of 14 ± 3 days in microcosms derived from treatment “–nZVI, –H2” Fig. , indicatDehalobacterium. Some strategies to attenuate such inhibitory effect include the surface modification of nanoparticles with surface stabilizers such as surfactants or polyelectrolytes, which slow down nZVI reaction rates, the production of H2, the release of ROS and iron ions, and avoid contact between nZVI particles and cells   Fig.  and D annm) Fig.  and E. Anm) Fig. . At the nm) Fig.  and F.FiL32 ratio values  biotic microcosms (control + H2), ii) biotic microcosms without H2 (control -H2), iii) abiotic-biotic microcosms containing 0.5 g nZVI/L, and iv) abiotic-biotic microcosms containing 1 g nZVI/L. Both control treatments removed 1,2-DCA at the same rate with the concomitant production of ethene, while those microcosms containing nZVI did not show 1,2-DCA depletion despite the circumneutral pH and the high production of H2 that could be used as electron donor for Dehalogenimonas .As shown in Fig. nas Fig. . Therefonas Fig. . This isnas Fig. , indicatDehalogenimonas activity by nZVI, our results are not necessarily transferable to field studies. The high complexity of the soil and groundwater composition could avoid the direct contact between nanoparticles and cells while providing optimum conditions for ORB growth .Despite the inhibition of Dehalobacterium and Dehalogenimonas was inhibited at 1 g nZVI/L, which was the optimal concentration of nZVI found to fully remove CF (500 μM). Further experiments discarded that changes in both pH and partial pressure of H2 provoked by nZVI exerted inhibition on Dehalobacterium. The fact that both Dehalogenimonas and Dehalobacterium recovered the dechlorinating capability after being transferred to nZVI-free medium pointed to soluble compounds released by particles as potential inhibitors. These findings have implication for remediation strategies aiming to combine both abiotic and biotic treatments to decontaminate aquifers with mixtures of chlorinated compounds. In particular, our study suggests that factors such as the buffering capacity of groundwaters and dilution processes can play crucial roles in the viability of this combined strategy in bioremediation treatments.Our laboratory tests demonstrated that dechlorinating activity of ESM 1(DOCX 236 kb)


As an emerging preparation strategy for MXenes, Lewis acidic etching has attracted increasing attention in the past few years benefiting from a series of merits.Lewis acidic etching method is mainly presented from etching mechanism, terminations regulation, in-situ formed metals and delamination of multi-layered MXenes.The applications of MXenes and MXene-based composites obtained by Lewis acidic etching route in energy storage and conversion, sensors and microwave absorption are carefully summarized. Since the discovery in 2011, MXenes have become the rising star in the field of two-dimensional materials. Benefiting from the metallic-level conductivity, large and adjustable gallery spacing, low ion diffusion barrier, rich surface chemistry, superior mechanical strength, MXenes exhibit great application prospects in energy storage and conversion, sensors, optoelectronics, electromagnetic interference shielding and biomedicine. Nevertheless, two issues seriously deteriorate the further development of MXenes. One is the high experimental risk of common preparation methods such as HF etching, and the other is the difficulty in obtaining MXenes with controllable surface groups. Recently, Lewis acidic etching, as a brand-new preparation strategy for MXenes, has attracted intensive attention due to its high safety and the ability to endow MXenes with uniform terminations. However, a comprehensive review of Lewis acidic etching method has not been reported yet. Herein, we first introduce the Lewis acidic etching from the following four aspects: etching mechanism, terminations regulation, in-situ formed metals and delamination of multi-layered MXenes. Further, the applications of MXenes and MXene-based hybrids obtained by Lewis acidic etching route in energy storage and conversion, sensors and microwave absorption are carefully summarized. Finally, some challenges and opportunities of Lewis acidic etching strategy are also presented. I. I3C2Tx d V2CTx) . In this3AlC2 MAX in dilute aqueous NaOH solution at 80 °C by Xie et al. in 2014 . . 3AlC2 M 10 days . The rea4N3Tx MXene in 2016, which opens the door to the preparation of MXenes via dry chemical etching techniques , , 2C and l safety . They al process . In 2021operties . Besidesfor 20 h . The ion2C nanosheets, which possess a thickness of few nanometers and a lateral size of around 100 µm 3C2Tx MXeate Fig. c–d, demonds Fig. e–f, facig−1 Fig. g, and re3C2Tx/CuS hybrids (T = –O and –Cl) via CuCl2·2H2O etching and following in-situ sulfurization treatment . . 3C2Cl2 ure Fig. b–e, whicg−1 Fig. f, and th+, Na+, K+, NH4+ and Mg2+)-intercalated Ti3C2Tx MXene, and a volumetric capacitance of 350 F cm−3 can be obtained in KOH electrolyte, which is better than that of traditional porous carbon. Later, freestanding Ti3C2Tx films prepared by LiF/HCl etching exhibit a high volumetric capacitance of 900 F cm−3 at 2 mV s−1 in H2SO4 electrolyte . . 3C2Cl2,3C2Cl2 MXene nanodots (QD-Ti3C2Cl2) via CdCl2 etching and subsequent hydrothermal reaction, and the nanodots are distributed on the surface of MXene nanosheets 3C2Clx MX3C2Clx quantum dots (Ti3C2Clx QDs) with an average size of around 8 nm were prepared through CuCl2 etching, subsequent hydrothermal reaction and ultrasonication treatment 0.90Pb(I0.83Br0.17)3 perovskite compound, which can also be confirmed by the peaks move in Pb 4f and Cl 2p XPS spectra. Additionally, scanning electron microscopy (SEM) images demonstrate that the perovskite grain size can be enlarged from 273 to 453 nm after the addition of Ti3C2Clx QDs , Ti3C2Cl2 MXene possesses an enlarged interlayer spacing, more active sites and stable architecture. Therefore, Ti3C2Cl2 electrode shows a smaller overpotential of 330 mV than HF-Ti3C2Tx MXene (390 mV) at 30 mA cm−2 for OER. Similarly, a lower HER overpotential of 259 mV can be obtained for Ti3C2Cl2 at 10 mA cm−2, while HF-Ti3C2Tx MXene demonstrates a large overpotential of 444 mV at the same current density. Notably, Ti3C2Cl2 electrode exhibits a low Tafel slope of 48 and 92 mV dec−1 for OER and HER, respectively, indicating excellent catalytic activity for water splitting.Water splitting provides a simple, effective and promising method for Hy crisis . In geney crisis . Neverth voltage . Explorisolution . Compare2, leading to a huge impact on the environment, such as greenhouse effect and the extinction of some species . . 3, as a.8% Fig. g. Additi3C2Cl2 MXene via ZnCl2 etching in 2021 and investigated its gas-sensing performance [3C2Cl2 MXene exhibits a response value of 13.2% to NH3, indicating the potential of Ti3C2Cl2 MXene in gas sensors. In addition, Au/Pt/Ti3C2Cl2 hybrids as sensors were fabricated by ZnCl2 etching and subsequent self-reduction process  hybrids via CoCl2 etching, and Ti3CNCl2(Co) composites were then distributed on the surface of reduced graphene oxide (RGO) nanosheets to form Ti3CNCl2-decorated RGO aerogel via the crosslinking effect of dopamine hydrochloride  such as MoAlB and Cr2AlB2, which is similar to MAX phase, have attracted intensive interests in the past few years. Therefore, the universal preparation of MBene by etching MAB precursors with Lewis acidic molten salts is worthy of in-depth investigation. Finally, the parameters of Lewis acidic etching reaction, including the molar ratio of MAX precursor and Lewis acidic salts, reaction temperature, time and atmosphere, are widely different in reported literatures  on the properties of various MXenes, such as electronic, optical and mechanical, magnetic properties as well as ion migration kinetics, hydrophilicity, chemical and thermal stability, etc.2 and Ti3C2Tx/FeS2. In the future, more studies should be performed to prepare MXenes/metal phosphides, MXenes/metal selenides or MXenes/metal tellurides hybrids via Lewis acidic etching route, which can be rationally and effectively applied in LIBs, SIBs, potassium-ion batteries and so on.So far, MXenes/metals hybrids prepared by Lewis acidic etching have been successfully applied in LIBs and SCs. In the future, MXenes/metals composites exhibit great research potential in catalysis, metal anodes and other fields. For example, benefiting from the unique layered structure and superior charge/mass transfer kinetics of MXenes as well as nucleation induction effect of some metals , MXenes/metals hybrids can be utilized as three-dimensional frameworks to suppress the dendrite growth of metal anodes . Furthermore, the obtained MXenes/metals hybrids can be transformed to MXenes/metal compounds composites via one-step sulfurization, phosphorization, selenization or tellurization treatment. Currently, most of the research was concentrated in MXenes/metal sulfides hybrids, such as N-MX-CoSn-BuLi in organic solvent NMF, there is still no safe, green, efficient and easily scalable method to delaminate the Lewis acidic molten salts-etched multi-layered MXenes. In the future, great efforts should be made to explore other appropriate delamination methods, such as high-energy ball milling and electrochemical delamination.Up to now, most of MXenes prepared by Lewis acidic etching are still at multi-layered state Table . The absIn this review, we have systematically introduced the Lewis acidic molten salts etching strategy from four aspects: etching mechanism, terminations regulation, in-situ formed metals and delamination of multi-layered MXenes. Briefly, the etching mechanism is mainly based on the replacement reaction between metal cations in Lewis acidic molten salts and zero-valence A-site atoms in MAX precursor. The difficulty degree of etching reaction mainly depends on the redox potentials of molten cations and A-site atoms, but has nothing to do with the M, X-site atoms and In conclusion, although some progress has been made in the development of Lewis acidic etching, it is still in the early stage. In order to solve the above-mentioned challenges, much more work should still be done in the near future. It can be anticipated that Lewis acidic etching will experience a rapid development period, which can attract more and more researchers in MXene community to widely utilize this emerging preparation strategy.


Staphylococcus aureus uses the Cnt system to secrete the metallophore staphylopine in zinc-limited host niches. Here, we show that staphylopine and the Cnt system facilitate bacterial copper uptake, potentiating the need for copper detoxification. During in vivo infection, staphylopine usage increased S. aureus susceptibility to host-mediated copper stress, indicating that the innate immune response can harness the antimicrobial potential of altered elemental abundances in host niches. Collectively, these observations show that while the broad-spectrum metal-chelating properties of metallophores can be advantageous, the host can exploit these properties to drive metal intoxication and mediate antibacterial control.Microorganisms can acquire metal ions in metal-limited environments using small molecules called metallophores. While metals and their importers are essential, metals can also be toxic, and metallophores have limited ability to discriminate between metals. The impact of metallophore-mediated non-cognate metal uptake on bacterial metal homeostasis and pathogenesis remains to be defined. The globally significant pathogen S. aureus to copper intoxication. In response to zinc starvation, S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria.During infection, bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes  Transition metals are essential for all forms of life. However, their bioavailability is limited in both the environment and host niches infected by pathogens. To overcome metal starvation, microbes from all three domains of life produce small metal-binding molecules or metallophores –3. WhiImmunological proteins such as transferrin, lactoferrin, and calprotectin (CP) reduce the availability of essential elements, including manganese (Mn), iron (Fe), and zinc (Zn) at sites of infection in an attempt to starve invaders , 13. To Staphylococcus, Pseudomonas, Yersinia, Paenibacillus, Serratia, Bacillus, and Vibrio -specific efflux pump CopA, which is induced in response to cytoplasmic accumulation of this metal . These observations indicate that activation of the Cu stress response is predominately dependent on the production and import of StP by the Cnt system.If metallophores contribute to Cu accumulation within the bacterial cytosol, it follows that the Cu stress response will be activated in environments that trigger the production of small opine molecules. In is metal . To evalis metal  was used ≥250 µM . In contnM CuSO4 . This in. aureus . To detet system , 24. Lose medium . Similarin ΔadcA . The latS. aureus was investigated. Wild-type S. aureus Newman and derivatives lacking AdcA and CntA were grown in the presence and absence of Cu in a Zn-limited medium. In the absence of Cu, all strains showed similar growth  ablated the growth impairment. To mimic the sequential Zn limitation followed by Cu exposure experienced during infection, the strains were grown in a Zn-limited medium and then spot-plated onto a solid medium with and without CuSO4. In the absence of Cu, wild type, ΔadcA, and ΔcntA were recovered equally. However, in the presence of 25 or 50 µM CuSO4 supplementation, ΔadcA showed a 10-fold greater sensitivity to Cu than wild type or ΔcntA in 60% (3/5) and 100% (5/5) of assays, respectively .Copper toxicity in bacteria can arise through direct modalities of action or indirectly through the disruption of essential metal uptake , 30. To µM CuSO4 . In the O4   . To evals tested . In the 4, but not 15 µM, resulted in ΔadcA accumulating less 66Zn than wild type or ΔcntA  enhanced the sensitivity of the ΔcopA mutant to Cu intoxication, whereas the use of the Adc system did not . Forcing S. aureus to rely on the StP-Cnt system further sensitized the bacterium to Cu intoxication, as ΔcopAΔadcA was 10-fold more sensitive to Cu than ΔcopA or ΔcopAΔcntA .As Cu negatively impacts both cellular processes and Zn uptake, we evaluated how the loss of Cu detoxification impacted these processes by assessing GAPDH activity and cellular metal accumulation . In the activity . In the  strains . Followi300-fold . NotablyopAΔcntA . The StPA strain , while iS. aureus to Cu intoxication. Compared to a metal-replete medium, treatment with CP resulted in a lower concentration of Cu being necessary to induce copA expression , yersiniabactin also binds and imports Cu into the periplasm and cytoplasm. This activity is thought to benefit UPEC  acquisition, can contribute to divalent cation acquisition, with yersiniabactin being necessary for the host , 54. In fit UPEC , 39. Howfit UPEC , 56. Fur species , 54, 57.The significance and biological impact of non-cognate metal-binding by metallophores have not previously been defined. While StP and, by extension, related metallophores provide crucial benefits to pathogens at the host-pathogen interface, this work establishes that the promiscuity of these small molecules can also render microbes susceptible to metal intoxication.S. aureus Newman and LAC derivatives were used for all experiments (S. aureus strains were grown at 37°C in either 5 mL tryptic soy broth (TSB) on a roller drum or on tryptic soy agar (TSA) plates for performing routine culturing or genetic manipulation. E. coli strains were routinely cultivated at 37°C in Luria broth with shaking or on Luria agar plates. For plasmid maintenance in E. coli and S. aureus, when appropriate, antibiotics were added at the following final concentrations: 100 µg/mL ampicillin, 50 µg/mL kanamycin, 10 µg/mL trimethoprim, 10 µg/mL chloramphenicol, and 1 µg/mL tetracycline. Both bacterial species were stored at −80°C in a growth medium containing 30% glycerol. The Newman ∆adcA, ∆cntA, ∆cntKLM, ∆copA, ΔcopAΔadcA, ΔcopAΔcntA, Δzur, ΔzurΔadcA, and ΔzurΔcntA strains were generated as previously described -containing vector pAH5 , 62S. autor pAH5 . All concopA and the cnt operon, S. aureus strains were grown overnight in chelex-treated RPMI plus 1% casamino acids (NRPMI) supplemented with 1 mM MgCl2, 100 µM CaCl2, 1 µM MnCl2 (4 in a 96-well round-bottomed microtiter plate and grown to mid-log phase (about 4 h) with orbital shaking (180 rpm) at 37°C. For assays using CP to impose a metal limitation, the overnight cultures were diluted 1:100 in 96-well round-bottom plates containing 100 µL of a growth medium, which consisted of 38% TSB and 62% CP buffer  in the presence or absence of 960 µg/mL of CP. For both assays using metal-defined medium and CP, the bacteria were harvested and resuspended in fresh NRPMI supplemented with 1 mM MgCl2, 100 µM CaCl2, and 1 µM MnCl2 at equivalent ODs. The bacteria were then diluted fivefold in 100 µL NRPMI supplemented with 1 mM MgCl2, 100 µM CaCl2, 1 µM MnCl2, and 10 µg/mL of chloramphenicol in the presence or absence of 10 µM of ZnSO4 and various CuSO4 concentrations in 96-well round-bottomed microtiter plates. The bacteria were incubated with orbital shaking (180 rpm) at 37°C, growth was measured by assessing optical density (OD600), and fluorescence  was assessed every hour using a BioTek Synergy H1 microplate reader.To assess the expression of µM MnCl2 , and 10 2, 100 µM CaCl2, and 1 µM MnCl2. They were then diluted 1:100 in 100 µL NRPMI supplemented with 1 mM MgCl2, 100 µM CaCl2, 1 µM MnCl2, and a range of CuSO4 concentrations and 100 µM ZnSO4 as indicated. Bacteria were incubated with orbital shaking (180 rpm) at 37°C, and growth was measured by assessing optical density (OD600) every 1–2 h using a BioTek Synergy H1 microplate reader. For spot plating assays using the defined medium, the strains were grown overnight in NRPMI supplemented with 1 mM MgCl2, 100 µM CaCl2, and 1 µM MnCl2  at 37°C. Following growth to an OD600 of ~0.5–0.6, cultures were serial diluted and spot plated on RPMI Medium 1640 (Gibco) agar (1%) plates containing various Cu concentrations.For liquid culture assays, overnight cultures were grown in 5 mL TSB in 15 mL conical tubes at 37°C on a roller drum. The overnight cultures were pelleted and resuspended in 5 mL NRPMI supplemented with 1 mM MgClµM MnCl2 . The nexS. aureus Newman strains was determined by inductively coupled plasma mass spectrometry (ICP-MS) essentially as previously described (600 of ~0.2) by centrifugation at 2,500 × g for 10 min, and washed two times with 0.1 M EDTA and then washed two further times with MilliQ water to remove adventitious trace element content. The cells were then suspended in 1 mL of MilliQ water, and an aliquot was collected for colony forming unit (CFU) determination. The bacteria were then centrifuged, and the supernatant was removed. Bacterial pellets were desiccated at 96°C overnight and then weighed to determine the dry cell mass of the pellet. The cellular material was digested in 250 µL of 65% (vol/vol) HNO3 at 96°C for 20 min. Insoluble material was removed by centrifugation at 20,000 × g for 25 min, and the supernatant was diluted in MilliQ-H2O to a final volume of 1 mL and analyzed in technical triplicate by ICP-MS on an Agilent 8900 ICP-MS/MS.The elemental content of the escribed , 69. SucS. aureus Newman strains were grown using the culturing parameters described for the copper susceptibility assays and harvested during the log phase (OD600 ~0.2). The cells were washed once with 50 mM sodium phosphate buffer (pH 7.5) containing 5 mM EDTA and then washed twice with 50 mM sodium phosphate buffer (pH 7.5) without EDTA. The cells were then resuspended in 500 µL of 50 mM sodium phosphate buffer (pH 7.5) and homogenized twice in a FastPrep-24 Bead beater at 6 m/s for 45 s with 5 min of incubation on ice in between. Cell lysates were centrifuged at 4°C in a microcentrifuge at 13,000 × g for 15 min. The protein concentration in the cell lysate was determined via BCA assay (Pierce). GAPDH activity in cell lysates was determined by adding 100 µL of cell lysate  to 100 µL of assay buffer  in a flat-bottomed 96-well plate. The reaction was followed by measuring absorbance at 340 nm every 2 min for 30 min on a BioTek Synergy H1 microplate reader. Negative controls  were included in the assay.For GAPDH assays, S. aureus strains were grown overnight in 5 mL TSB in 15 mL conical tubes at 37°C on a roller drum. The overnight cultures were diluted 1:50 in fresh 10 mL TSB in 50 mL conical tubes and grown to early log phase (OD600 ~ 0.4) at 37°C in a shaking incubator at 180 rpm. Bacteria were centrifuged at 4,000 rpm for 10 min and resuspended in phosphate-buffered saline (PBS) to a concentration of 1 × 109 CFU/mL. Each bacterial competing pair, containing distinct antibiotic markers, was mixed in a 1:1 ratio and placed on ice. The flanks of ten-week-old female C57BL/6 mice were shaved and depilated with Nair before infection. Mice were injected subcutaneously with 50 µL of 1 × 109 CFU/ml of S. aureus mutant pairs (containing either kanamycin or chloramphenicol antibiotic markers) as specified. The infection was allowed to proceed for 7 or 14 days, after which the mice were sacrificed. The skin abscesses were harvested and homogenized in PBS. Bacterial burdens were enumerated by plating serial dilutions on appropriate antibiotic-containing TSA plates. CIs were calculated. CI is defined as the bacterial output ratio divided by the bacterial input ratio used to initiate the infection.All animal experiments were performed as previously described , 70. S. All statistical analyses were performed using GraphPad Prism version 9 and the indicated statistical tests.


Background and Objectives: Psoriasis is an immune-mediated chronic inflammatory skin disorder and commonly associated with highly noticeable erythematous, thickened and scaly plaques. Deubiquitinase genes, such as tumor necrosis factor-alpha protein 3 , the cylindromatosis (CYLD) and Cezanne, function as negative regulators of inflammatory response through the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. In this study, polymorphisms and expressions of A20, CYLD and Cezanne genes as well as immunophenotype in psoriatic patients were determined. Materials and Methods: In total, 82 patients with psoriasis and 147 healthy individuals with well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by direct DNA sequencing, gene expression profile by quantitative real time-polymerase chain reaction (PCR), immunophenotype by flow cytometry, and the secretion of cytokines and cancer antigen (CA) 125 by enzyme-linked Immunosorbent assay (ELISA). Results: The inactivation of A20, CYLD and Cezanne and increased levels of TNF-α, IFN-γ and CA 125 was observed in psoriatic patients. Importantly, patients with low A20 expression had significant elevations of triglyceride and total cholesterol concentrations and higher numbers of CD13+CD117− and CD19+CD23+ (activated B) cells than those with high A20 expression. Genetic analysis indicated that all rs4495487 SNPs in the JAK2 gene, rs200878487 SNPs in the A20 gene and four SNPs  in the Cezanne gene were associated with significant risks, while the rs10974947 variant in the JAK2 gene was at reduced risk of psoriasis. Moreover, in the Cezanne gene, p.W433R was predicted to be probably damaging by the Polyphen-2 prediction tool and an AA/CC haplotype was associated with a high risk of psoriasis. In addition, patients with higher CA 125 levels than the clinical cutoff 35 U/mL showed increased levels of IFN-γ than those with normal CA 125 levels. Conclusions: A20 expression was associated with lipid metabolism and the recruitment of CD13+ CD117− and activated B cells into circulation in psoriatic patients. Besides this, the deleterious effect of the p.W433R variant in the Cezanne gene may contribute to the risk of psoriasis. The mospatients ,4,5. Thepatients , which cpatients . The actpatients , while Cpatients . CD23 ispatients .Immunogenetic investigations revealed that most psoriasis susceptibility loci are linked to inflammatory response-related genes. Among them, deubiquitinase (DUB) genes, including tumor necrosis factor α-induced protein 3 , tumor suppressor cylindromatosis (CYLD) and Cezanne, play important roles in deubiquitinating target proteins by cleaving their polyubiquitin chains to suppress the activation of downstream signalling pathways. The attenuated expression of A20 and CYLD is shown in patients with leukemia/lymphoma ,14,15 anSimilar to A20, the association of JAK2 with psoriasis susceptibility has also been extensively studied, as JAK2 is involved in the control of cellular growth and proliferation during immune responses in these patients . A20 is In this study, the expressions of A20, CYLD and Cezanne were examined by quantitative real time-PCR and immunophenotype by flow cytometry in 82 patients with psoriasis and 147 healthy individuals. Besides this, polymorphisms of A20, Cezanne, CYLD and JAK2 genes were also determined by direct DNA sequencing.A total of 82 untreated psoriatic patients and 147 healthy volunteers used as controls were recruited into the study at the Thien Phu Duong Traditional Medical Clinic, Hanoi, Vietnam. The diagnosis of psoriasis was based on the 2016 WHO criteria , includiTotal mRNA was isolated using the Qiashredder and RNeasy Mini Kit from Qiagen according to the manufacturer’s instructions. For cDNA first strand synthesis, 1 µg of total RNA in 12.5 µL DEPC-H2O was mixed with 1 µL of oligo-dT primer  and heated for 2 min at 70 °C. To determine the transcript levels of A20, Cezanne, CYLD and GAPDH, quantitative real-time PCR with the LightCycler System  was applied. The following primers were used: A20 primers—5′-TCCTCAGGCTTTGTATTTGA-3′ (forward) and 5′-TGTGTATCGGTGCATGGTTTT-3′ (reverse); Cezanne primers—5′-ACAATGTCCGATTGGCCAGT-3′ (forward) and 5′-ACAGTGGGATCCACTTCACATTC-3′ (reverse); CYLD primers—5′-TGCCTTCCAACTCTCGTCTTG-3′ (forward) and 5′-AATCCGCTCTTCCCAGTAGG-3′ (reverse), and GAPDH primers—5′-GGAGCGAGATCCCTCCAAA-3′ (forward) and 5′-GGCTGTTGTCATACTTCTCAT-3′ (reverse). PCR reactions were performed in a final volume of 20 µL containing 2 µL cDNA, 2.4 µL MgCl2 (3 µM), 1 µL primer mix (0.5 µM of both primers), 2 µL cDNA Master SybrGreen I mix , and 12.6 µL DEPC-treated water. The target DNA was amplified during 40 cycles of 95 °C for 10 s, 62 °C for 10 s, and 72 °C for 16 s, each with a temperature transition rate of 20 °C/s, a secondary target temperature of 50 °C, and a step size of 0.5 °C. Melting curve analysis was performed at 95 °C for 0 s, 60 °C for 10 s and 95 °C for 0 s to determine the melting temperature of primer dimers and the specific PCR products. The ratio between the respective gene and corresponding GAPDH was calculated per sample according to the ∆∆ cycle threshold method .Genomic DNA was isolated from peripheral blood samples using a DNeasy blood and tissue kit . To determine polymorphisms of the JAK2, A20, CYLD and Cezanne genes, polymerase chain reaction (PCR) and DNA sequencing  were performed as previously described . The Gen5) were incubated in 100 µL FACS buffer (PBS plus 0.1% FCS) containing fluorochrome-coupled antibodies to CD45, FoxP3, CD19, CD13, CD23, CD117, CD3, CD4, and CD25  at a concentration of 10 µg/mL. After incubating with the antibodies for 60 min at 4 °C, the cells were washed twice and resuspended in FACS buffer for flow cytometry analysis.Immunophenotypic features of PBMCs from psoriatic patients and healthy volunteers were determined by flow cytometry  as previously described . Cells  according to the manufacturer’s protocol.https://www.ncbi.nlm.nih.gov/) accessed on 10 January 2022. The information for SNP ID of these genes was retrieved from the NCBI’s SNP database (https://www.ncbi.nlm.nih.gov/snp/) accessed on 6 January 2023. Bioedit software was used for the initial analysis of the sequences. To analyze the functional consequences of deleterious SNPs, a PolyPhen2 program (http://genetics.bwh.harvard.edu/pph2/index.shtml) accessed on 20 May 2023 was used. The PolyPhen-2 score varied from 0.0 (tolerated) to 1.0 (deleterious), in which the SNPs were designated “probably damaging”, “potentially damaging”, “benign” or “unknown”.Data of genes related to this record were collected from NCBI (2) tests, crosstabs and odds ratios were used for dichotomous data, and the Mann–Whitney U test for nominal data. p < 0.05 was considered statistically significant.Statistical analysis was performed with the SPSS version 20  and GraphPad Prism version 8.4 softwares . To analyze the data, Chi-squared  expressing cells in the patients with low A20 expression (+ cells is found in atopic dermatitis and psoriasis [+CD3+CD4+CD25+FoxP3+- (regulatory T), CD45+CD19+CD25+- (activated B), CD45+CD3+CD25+- (activated T) and CD13+CD117+-expressing cells were unaltered compared to healthy controls (data not shown). The results suggest that A20 expression in psoriatic patients was related to lipid metabolism, and the expansion of CD13+CD117−- and CD23+CD19+-expressing cells into circulation.Finally, immunophenotype in psoriatic patients was determined by flow cytometry analysis. We found the expansion of CD13pression E,F, as cp > 0.05)  . Importa > 0.05) , while t > 0.05) .p > 0.05)  . Moreovep > 0.05) (The sequencing of the CYLD gene identified that an SNP (c.2351-118) in intron 14 and two SNPs (c.2483+53 G>A and c.2483+188 G>A) in intron 15 were found . The gen > 0.05) .p. W433R), in exon 11 were detected in this gene, in which the SNPs rs1030371296 C>T and c.1642 T>A (p.W433R) were non-synonymous SNPs (nsSNPs), causing changes in the amino acid residues, and a remaining SNP rs782178516 C>T was silent  were unidentified SNPs and the two remaining intronic SNPs (rs587631702 A>T and rs1230581026 C>T) are reported in NCBI’s SNP database A. Moreovs silent A. The geAfter the determination of susceptibility to psoriasis by evaluating the deleterious effects of the nsSNPs on the Cezanne gene, p.W433R was predicted to be probably damaging by Polyphen-2 with a score of 1  B. Accordp < 0.001)  . The linIn this study, we observed for the first time that the expressions of A20, CYLD and Cezanne were inactivated in psoriatic patients. In agreement, A20 prevents inflammatory reactions in psoriatic arthritis-like disease , and theTo examine genetic alterations, we observed that the detected frequency of the CG genotype of rs200878487 in the A20 gene was significantly higher in the patient group compared to healthy individuals. Recently, two A20 variants, including rs582757 and rs6918329, have been identified as causal SNPs in psoriasis , and the−/− mice do not exhibit skin abnormalities [Next, CYLD is prominently expressed in the epidermis; however, CYLDmalities . Recentlmalities . In thisUnlike A20 and CYLD, associations between polymorphisms in the Cezanne gene and autoimmune diseases are not well documented. Among the 11 SNPs identified in the Cezanne gene, we found that 4 SNPs, including the AC genotype of c.1584-375, AC genotype of c.1584-374, CT genotype of rs1230581026 and TA genotype of p.W433R, left one at increased risks of psoriasis. Differently, our recent study indicated that the c.1584-287 SNP in this gene is involved with the risk of polycythemia vera [Functional studies of the A20 gene have demonstrated that A20 plays important roles in regulating pro-inflammatory gene expression through the activation of intracellular signaling pathways such as JAK2 . Here, t+CD117−- and CD23+CD19+-expressing cells in patients with a low A20 expression as compared to those with a high A20 expression and healthy controls. Consistently, CD13 is up-regulated on endothelial cells at sites of inflammation [+CD117− and CD23+CD19+ cells in the circulation in patients with psoriasis were revealed for the first time. Recently, B cell-activation plays important roles in different pathological stages of psoriasis, and is correlated with disease severity [+CD4+ and CD25+ T cells were found in four subjects, with an incidence of 4.88%, whereas the numbers of these cells in other patients were unchanged (data not shown). Previously, the activation of CD4 T cells in the peripheral blood of psoriatic patients has been determined to cause prolonged inflammation [For the determination of immunophenotypic features, we observed the expansion of CD13ammation , and oveammation . The inhammation . Howeverseverity ,6, whereseverity . We addiSimilarly to a recent study , we obseLastly, the levels of CA125 were significantly increased in the patient group. Recently, CA125 has been found to be constitutively expressed in the skin of psoriatic patients , and its+ CD117− and activated B cells into circulation, in psoriatic patients. Moreover, the deleterious effects of the p.W433R variant in the Cezanne gene may contribute to the risk of psoriasis, making it a good object of further study regarding its role in regulating the functional activation of immune cells in psoriatic patients.A20 levels have been found to be associated with lipid metabolism, and the recruitment of CD13


Waxy maize, mainly consumed at the immature stage, is a staple and vegetable food in Asia. The pigmentation in the kernel of purple waxy maize enhances its nutritional and market values. Light, a critical environmental factor, affects anthocyanin biosynthesis and results in pigmentation in different parts of plants, including in the kernel. SWL502 is a light-sensitive waxy maize inbred line with purple kernel color, but the regulatory mechanism of pigmentation in the kernel resulting in purple color is still unknown.In this study, cyanidin, peonidin, and pelargonidin were identified as the main anthocyanin components in SWL502, evaluated by the ultra-performance liquid chromatography (UPLC) method. Investigation of pigment accumulation in the kernel of SWL502 was performed at 12, 17, and 22 days after pollination (DAP) under both dark and light treatment conditions via transcriptome and metabolome analyses.4CL2, CHS, F3H, and UGT, was reduced under dark treatment. Dynamic changes were identified in genes and metabolites by time-series analysis. The genes and metabolites involved in photosynthesis and purine metabolism were altered in light treatment, and the expression of genes and metabolites associated with carotenoid biosynthesis, sphingolipid metabolism, MAPK signaling pathway, and plant hormone signal transduction pathway were induced by dark treatment. Light treatment increased the expression level of major transcription factors such as LRL1, myc7, bHLH125, PIF1, BH093, PIL5, MYBS1, and BH074 in purple waxy maize kernels, while dark treatment greatly promoted the expression level of transcription factors RVE6, MYB4, MY1R1, and MYB145.Dark treatment affected genes and metabolites associated with metabolic pathways of amino acid, carbohydrate, lipid, and galactose, biosynthesis of phenylpropanoid and terpenoid backbone, and ABC transporters. The expression of anthocyanin biosynthesis genes, such as This study is the first report to investigate the effects of light on waxy maize kernel pigmentation and the underlying mechanism at both transcriptome and metabolome levels, and the results from this study are valuable for future research to better understand the effects of light on the regulation of plant growth. Zea mays L.) is one of the main fresh-eating maize types with many excellent characteristics in terms of starch composition and economic value. Purple waxy maize possesses high nutritional and economic value because it is rich in anthocyanins , cinnamic acid 4-hydroxylase (ZmC4H), and 4-coumarate CoA ligase (Zm4CL) are genes involved in the phenylpropanoid pathway, inducing the first enzymatic step in the flavonoid pathway (a1) (a2) (bz1) (aat1) (bz2) (red1 (r1) and booster1 (b1) which belong to the basic helix-loop-helix (bHLH) transcription factor family, colorless1 (c1), purple plant1 (pl1) and pericarp color1 (p1) of the MYB-like transcription factors, and the WD40 factor pale aleurone color1 (PAC1)  , anthocya1) (a2) , anthocy2) (bz1) , anthocy) (aat1) , and glu1) (bz2) . The reg1 (PAC1) . The exp1 (PAC1) .CHS, CHI, DFR, F3H, LDOX, and F3’5’H) and regulatory genes   . The mecto light . In addito light . AlthougIn this study, we investigated photosensitive waxy maize inbred line SWL502, which synthesized a significantly decreased amount of total anthocyanin with bagging, through a large number of bagging screenings. Here, we analyzed the transcriptomic and metabolomic data of bagged and unbagged waxy maize kernels and identified system-wide alterations in metabolites, structural genes, and regulation in response to light and dark treatments. This is the first in-depth look at how light affects the pigmentation of purple waxy maize kernels, and it paves the way for future studies and genetic manipulations of waxy maize kernel coloration.The purple waxy maize inbred line SWL502 was grown in the summer of 2021 at the experimental station of Shanghai Academy of Agricultural Sciences, Shanghai, China. After pollination, the whole ears were covered with foil paper bags for dark treatment (D) which were completely light‐impermeable, on June 16th, 2021. Meanwhile, unbagged ear samples, which were grown under natural light (L) conditions, served as controls. For each treatment, kernels from the middle part of three ears were sampled, mixed, and randomly divided into nine biological replicates: three for RNA-seq, and six for metabolome analysis. Kernel samples were collected at 12, 17, and 22 days after pollination (DAP), which were immediately frozen in liquid nitrogen and stored at -80°C for storage after sampling.2+b*2)0.5. Hue value measures the most obvious value of a color and was calculated by using arctan (b*/a*). The color index of red grapes (CIRG) was calculated with the formula CIRG = (180-h)/(L∗ + C)  in the L* a* b* color space (CIELAB). Chroma, an expression of the purity or saturation of a single color, was calculated by using (a*(L∗ + C) . Saturat-1 at 40 °C. The wavelength was set at 530 nm. Delphinidin (CAS: 528-53-0), cyanidin (CAS: 528-58-5), petunidin chloride (CAS: 1429-30-7), pelargonidin (CAS: 134-04-3), peonidin (CAS: 134-01-0), and malvidin (CAS: 643-84-5) were used as standards for anthocyanins. All samples were analyzed in six biological triplicates.Anthocyanin mixtures from the waxy maize kernels were extracted and quantified according to the agricultural standard of China (NY/T 2640-2014) with modification. In brief, frozen waxy maize kernels were ground into powder in liquid nitrogen. Approximately 1 g of the maize powder was added in 10 mL of the ethanol-water-HCl mixture  for ultrasonic extraction for 30 minutes, boiling water bath for 1 h, and followed by centrifugation to collect the supernatant. The supernatant was then passed through a 0.45 μm membrane filter , and analyzed by ultra-performance liquid chromatography (UPLC) using a BEH C18 analytical column . A quantitative analysis of anthocyanin compounds was performed by an Acquity I-Class UPLC system with an ultraviolet detector  using a formic acid-acetonitrile mixture  and formic acid-acetonitrile mixture  of the following gradient: 0 min, 10% A; 5 min, 18% A; 6 min, 18% A; 8 min, 10% A. The flow rate was 0.4 ml minTotal RNA was extracted using the TRIzol reagent  according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer . RNA integrity was assessed using the Agilent 2100 Bioanalyzer . Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit  according to the manufacturer’s instructions. The libraries were sequenced by an Illumina Novaseq 6000 platform in OE Biotech Co., Ltd. .Raw reads were processed using FASTQC and Trimmomatic  to obtai-1 throughout the course. The column oven was adjusted to 45°C, and the injection volume was 2 μL. Data were collected in both positive and negative ion modes.A total of 80 mg of freeze-dried maize kernels were mixed with 800 μl of methanol-water  and crushed for 2 min at 60 Hz using a mixer mill , followed by ultrasonic extraction in an ice-water bath for 30 min, and extracted overnight at -20°C. After centrifugation at 13000 rpm and 4°C for 10 min, the supernatant was filtered using a 0.22 μm organic phase pinhole filter. The filtrate was then transferred to an LC injection vial and stored at -80°C until LC-MS analysis. Using a UHPLC-ESI-MS/MS system, the sample extracts were examined . The analysis was conducted using an ACQUITY UPLC HSS T3 column . The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Using a gradient program of 95% A and 5% B as the initial condition, sample measurements were conducted. Within 16 minutes, a linear gradient of 5% A and 95% B was programmed, which was maintained for 1 min. The flow rate was maintained at 0.35 mL·min-1, methanol as the dissolving agent). The tube was added with 360 μL of precooled methanol and put in the refrigerator at -20°C for 5 min. After grinding  with steel balls, the powder was extracted by ultrasonic for 30 min at 0°C. Then 200 μL of chloroform and 400 μL of water were added to the powder and mixed evenly. After ultrasonic extraction in ice water for 30 min, the solution was placed at -20°C for another 30 min, followed by centrifugation for 10 min at 13 000 rpm at 4°C. Afterwards, 150 μL of the supernatant was transferred into a glass vial and dried by a centrifugal dryer. The obtained dried powder was mixed with 80 μL of methoxyamine hydrochloride pyridine solution (15 mg·mL-1). After vortex shaking for 2 min, the oximation reaction was carried out in a 37°C shaking incubator for 90 min. The sample was then added with 50 μL of BSTFA (including 1%TMCS) as the derivatization reagent, 20 μL of n-hexane, and 10 μL of ten types of internal standards . After vortex shock for 2 min, the reaction was set for 60 min at 70°C. Finally, GC-MS analysis was performed at room temperature for 30 min. The obtained sample extracts were examined using DB-5MS column  with parameters set as follows: carrier gas, high purity helium; flow rate, 1.0 mL·min-1; and injection port temperature, 260°C. The injection volume was 1 μL, and the solvent delay was 5 min without shunt injection. Programmed heating was set as follows: the initial temperature of the column incubator was 60°C, holding for 0.5 min, then with a heating rate of 8°C min-1 to 125°C, 8°C min-1 to 210°C, 15°C min-1 to 270°C, 20°C min-1 to 305°C, maintaining for 5 min. Mass conditions were as follows: electron impact (EI); full scan mode with a range of 50-500 m; ion source temperature at 230°C; MS quard temperature at 150°C; electron energy at 70 eV. Quality control samples were prepared by mixing equal volumes of the extracts of all samples.A total of 80 mg of maize kernels were placed in a 1.5 mL tube with 20 μL of internal standard  was used to extract the variable importance in the projection (VIP) values from the OPLS-DA results, including score graphs and permutation plots. To prevent overfitting, a permutation test was performed 200 times. Differential metabolites consisting of metabolites with VIP > 1 and absolute Log2FC >= 1 were chosen for further study. The annotated metabolites were linked to the KEGG database. Pathways with substantially upregulated metabolites were then screened in MetaboAnalyst (v3.0), and their significance was established using the p values of the hypergeometric test. The Mfuzz (v 2.56.0) package was used for all trend analyses.The original LC-MS data were processed by the software Progenesis QI V2.3  for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization to obtain the data matrix. The unsupervised PCA of LC-MS and GC-MS data after unit variance scaling was carried out by using the Actin was used as an internal control to calculate relative gene expression in this study. The gene expression for each sample was measured by qRT-PCR using the relative quantification method . The reaction was carried out in qTOWER 2.2 Quantitative Real-Time PCR Thermal Cyclers (Germany). The cycling conditions were 3 min of predenaturation at 95°C, then 95°C for 10 s, 60°C for 30 s, and 72°C for 90 s (39 cycles). The housekeeping gene n method . PrimersThe effect of light on pigmentation in SWL502 kernel was investigated, and the findings revealed that there was no significant difference in kernel color between bagged and unbagged ears at 12 DAP. However, the color of kernels differed dramatically at 17 and 22 DAP, indicating that 12 to 22 DAP is a crucial period for pigment formation in SWL502  transcription complex, which is a crucial transcriptional regulator in the flavonoid pathway in the plant. Overall, 21 bHLH, 18 MYB, and 13 WD40 TFs were differentially expressed between light and dark conditions. The expressions of seven In addition, seven TF families  were also identified as important regulators involved in light response  can be found in HZ and YL conceived the project and designed the experiments. YY, YFX, AK, CY, CW, and CZ performed the experiment and YL wrote the manuscript. Validation was done by WG, HW, YH, PS, and YG. Formal analysis was performed by WS, BB, and BS. The writing—review, and editing were done by XZ, YBX, and PB. All authors contributed to the article and approved the submitted version.


Franz diffusion cell experiments were performed using mice with full-thickness skin. At various times over 24 h, 300 μL of the receptor were withdrawn and replaced with fresh medium. Caffeine was analyzed spectrophotometrically at 272 nm. The skin irritation effects of the hydrogels applied once a day for 21 days were investigated in mice. The steady-state flux (JSS) of the caffeine hydrogel was 30 ± 19.6 µg cm−2 h−1. An increase in caffeine JSS was induced by Lippia origanoides > Turnera diffusa > eugenol > carvacrol > limonene, with values of 150 ± 14.1, 130 ± 47.6, 101 ± 21.7, 90 ± 18.4, and 86 ± 21.0 µg cm−2 h−1, respectively. The Kp of caffeine was 2.8 ± 0.26 cm h−1, almost 2–4 times lower than that induced by Lippia origanoides > Turnera diffusa > limonene > eugenol > carvacrol, with Kp values of 11 ± 1.7, 8.8 ± 4.2, 6.8 ± 1.7, 6.3 ± 1.2, and 5.15 ± 1.0 cm h−1, respectively. No irritating effects were observed. Lippia origanoides, Turnera diffusa, eugenol, carvacrol, and limonene improved caffeine’s skin permeation. These compounds may be as effective as the PE in TDD systems.The study aimed to determine the enhanced effects of essential oils (EOs) and plant-derived molecules (PDMs) as penetration enhancers (PEs) for transdermal drug delivery (TDD) of caffeine. A 1%  Overcoming the skin barrier (or certain layers of the skin) for drug entry without deleterious skin reactions  is still a major challenge to resolve. The skin is a complex structure formed by epidermal, dermal, and hypodermal layers that acts as a remarkably effective barrier to the entrance of exogenous agents, including drugs. Its barrier function is due to the stratum corneum (SC), the most superficial layer of the skin, described as a “brick-and-mortar” structure consisting of tightly arranged corneocytes enveloped by an intercellular lipid matrix . TransdeThe focus has been on identifying effective penetration enhancers (PE) from natural sources. For example, essential oils and terpenes are one of the encouraging groups of candidates to be used as clinically acceptable PE ,5. EssenAs part of a research program based on the discovery of multifunctional biological activities of Colombian essential oils and their constituents, in this case, those related to the improvement of some skin conditions that occur within the deeper layers of the skin and internal organs, this study aimed to determine the enhancing effect of EOs and PDMs obtained from Colombian plants as skin PEs of caffeine , a hydroLippia origanoides (Verbenaceae) plants (0.5–1.2%). EO chemical characterization was performed by GC/MS with electron ionization  and both polar and nonpolar capillary columns, using many standard compounds as well for confirmatory identification. In L. origanoides (Verbenaceae) and Turnera diffusa (Passifloraceae) plants, and L. origanoides chemotypes were differentiated according to their main compounds, as follows: (1) Phellandrene-chemotype ; in these EOs, α- and β-phellandrenes with trans-caryophyllene and its oxide prevailed; (2) Carvacrol-chemotype (EO8); and (3) Thymol-chemotype (EO19); in the latter EOs, the amount of phenolic compounds  represented more than 50–70% of the total composition. Sesquiterpene hydrocarbons were the main constituents of the Turnera diffusa EO, while monoterpene hydrocarbons and their oxygenated derivatives dominated the EOs distilled from Steiractinia aspera (EO1), Calycolpus moritzianus (EO4), Piper anduncum (EO5), and phellandrene-chemotype L. origanoides EOs .The essential oils studied here were obtained by microwave-assisted hydrodistillation from eight aromatic plants cultivated at the Agroindustrial Pilot Complex and Botanical Garden  at CENIVAM . The EO yields varied from 0.1 to 1.2% . The higp < 0.05) than that of gels with caffeine alone . The average pH was 5.5 and stable for up to 30 days, except for caffeine plus PDM22, which slightly decreased from 5.5 to 4.5 after 7 days. Since natural skin surface pH is acidic on average  [® as a gelling agent was an important achievement; it is a crosslinked polyacrylic polymer that is acidic but neutralized with an appropriate base  to achieve its thickening property. Carbopol-hydrogels are three-dimensional polymer networks that are biocompatible, adhesive, and biodegradable and can absorb large amounts of water and load large amounts of water-soluble drugs. In addition, similar to smart hydrogels, they can control drug release by responding to stimuli such as pH, temperature, and light [Hydrogels of caffeine alone and with EO or PDM were easily prepared . All forbelow 5) , preparebelow 5) ,16. The nd light ,16. Sincnd light .−2) of caffeine that permeates across the skin throughout the study (0–24 h) without EO or PDM had an average of 619.35, SD = 236,56 µg cm−2 , with a steady-state flux (JSS) of 32.28 ± 3.25 µg cm−2 h−1 (n = 12) (The cumulative amount (µg cm(n = 12) . Caffeinw/w EO on the cumulative amount of caffeine that permeates across the skin and the resulting skin permeation parameters are shown in Lippia origanoides, Thymol chemotype) and EO2 (Turnera diffusa) significantly enhanced the Jss values and permeability coefficients (Kp) with an enhancement index (EI) of 3.7 and 3.0, respectively. In contrast, the lowest enhancement in caffeine flux was obtained with EO1 (Steiractinia aspera), EO4 , and EO5 (Piper anduncum) with EI values lower than 0.9. Lippia origanoides H.B.K. (Verbenaceae) is an aromatic plant growing in different Colombian regions and used in traditional medicine due to its antioxidant, anti-viral, anti-genotoxic, anti-parasitic, and repellent activities [p-cymene > carvacrol > β-myrcene and γ-terpinene  (Passifloraceae) plant. It is a medicinal plant used traditionally as an aphrodisiac, especially in Central America, and is effective against diabetes and gastric, skin, and respiratory diseases [T. diffusa-EO as a PE; however, some PE activities of valencene and other sesquiterpenes such as farnesol and neridol have been described for drugs such as 5-fluorouracil , propranolol hydrochloride , and valsartan  [T. diffusa EO main constituents have been related to interesting activities such as anesthetic (dehydrofukinone) and anti-cancerogenic (β-elemene) activities and acting as a precursor of phytoalexins such as capsidiol (5-epi-aristolochene) [The other promising EO was distilled from the diseases . In contogP 4.5) ,23. To dlochene) ,25,26.w/w PMD are shown in p < 0.05) increase in caffeine flux compared with caffeine gel alone. No significant difference was found between the mean values of lag time. Penetration from caffeine gels containing PMD was greater than that of the control, displaying an EI higher than 1 . Some carvacrol formulations containing 10% carvacrol in propylene glycol or 3 mM carvacrol in PBS enhanced testosterone  and corticosterone  permeate through human skin by 7 to 10 times. In addition, a high enhancement (approximately 38 times) of azidothymidine  and increased flow and coefficient of permeability for haloperidol  in formulations containing 5% carvacrol/alcohol have been reported ,34,35. CLippia , Origanutivities . No avaiFinally, limonene was also an important caffeine PE. Limonene is one of the most studied drug-enhancer terpenes . A dose-L. origanoides chemotypes, we demonstrated the skin irritancy of 100–20% thymol- and carvacrol-chemotypes (but not phellandrene) used topically but used at 10% or less, and any change both macro and at the histopathological level was detected [In this work, mice showed no irritation signs or skin edema after treatment. Carbopol hydrogels containing caffeine alone or caffeine plus EO19, EO2, PDM6, PMD19, and PMD22 at 1% were safe. The treated skin was intact; no inflammation or erythema compared to the untreated site was observed . In a previous study, using different detected . The usedetected . HoweverSteiractinea aspera (EO1), Turnera diffusa (EO2), phellandrene- , carvacrol- (EO8), and thymol- (EO19) Lippia origanoides chemotypes, Calycolpus moritzianus (EO4), and Piper anduncum (EO5) aromatic Colombian plants. The ex situ plant collection was kept under controlled agricultural conditions , and only healthy and undamaged plants, previously chopped, were used immediately for their distillation. A contract for access to genetic resources and derived products for bioprospecting purposes, No. 270, was signed between the Ministry of Environment and Sustainable Development and Universidad Industrial de Santander.Eight EOs were distilled at the CENIVAM Experimental Agro-Industrial pilot complex, Universidad Industrial de Santander, Colombia, from −1 constant flow. The GC oven temperature was programmed as follows: from 45 °C (5 min) to 150 °C (2 min) at 4 °C/min, then to 300 °C (10 min) at 5 °C/min for the nonpolar DB-5MS column, and from 50 °C (5 min) to 150 °C (7 min) at 4 °C/min, then to 230 °C (50 min) at 4 °C/min) for the polar DB-WAX column. All experimental data  were processed using MSD ChemStation G1701DA software .Essential oil extraction was performed by microwave-assisted hydrodistillation using Clevenger equipment adapted to a microwave system as described elsewhere . The majCaffeine, citronellal, limonene, bromonaphthalene, carvacrol, eugenol, α-phellandrene, trans-β-caryophyllene, carbopol 940, sodium benzoate, and triethanolamine were purchased from Sigma-Aldrich . All the reagents were of analytical grade. The code, structure, molecular weight (MW), and LogP of the PDMs are shown in w/w) of Carbopol 934 in distilled, deionized water. Caffeine (1.0% w/w), sodium benzoate (0.1%), and EO or PMD at 1.0% w/w were added. The stirring was continued for 30 min and thereafter neutralized using 0.5% triethanolamine until a transparent gel appeared. The gels were inspected visually for their color, homogeneity, consistency, and phase separation. The pH was measured using a digital pH meter, and the viscosity was measured using a viscometer . The final concentration of caffeine was analyzed by UV-Vis spectrophotometry at 272 nm. For a calibration curve, a standard stock solution of caffeine was prepared by dissolving 100 mg of caffeine in 100 mL of distilled water and diluting 1:10 with distilled water (100 µg/mL). Standard solutions of 1–50 µg/mL of caffeine in distilled water were read at 272 nm, and the absorbance calibration curve was plotted (concentration versus absorbance). The correlation coefficient was 0.99 (y = 0.0425x + 0.1343).Hydrogels were prepared with constant stirring by dispersing 1.0% .BALB/c mice were trichotomized, and after 24 h, they were anesthetized by intraperitoneal injection of a ketamine/xylazine cocktail and euthanized by cervical dislocation. Dorsal full-thickness skin was obtained, and the subcutaneous tissue was carefully removed and washed in phosphate-buffered saline (PBS) with a pH of 7.4 twice. Skin samples were wrapped in aluminum foil and stored in a freezer at −20 °C until use, not exceeding 3 months. On the day of the experiment, the skin was left at room temperature and cut into pieces, and skin samples were mounted over the Franz cells with the SC side facing the donor compartment.2 and a fluid receptor of PBS pH 7.4 were used. The receptor compartment of 3 mL PBS at pH 7.4 was continuously mixed using a magnetic stirring bar (300 rpm) at 32 °C. Three hundred milligrams of caffeine alone, caffeine plus EO, or PMD gels  were placed on the membrane surface in the donor compartment and sealed with parafilm to avoid evaporation. Samples of 300 µL were withdrawn at 0, 1, 2, 4, 6, and 24 h from the receptor phase and immediately replaced with the same volume of the receptor fluid. The amount of caffeine was analyzed spectrophotometrically at 270 nm by the validated UV-Vis method described previously. The integrity of the mouse membrane was examined after the experiment. Skin membranes were fixed using 10% buffered (phosphate buffer) formalin and embedded in paraffin blocks. Sections (5 µm in thickness) were stained with hematoxylin-eosin (H–E) and examined microscopically.Franz diffusion cells  with a diffusional effective area of 0.196 cm−2), steady-state flux , permeability coefficient (cm h−1), and lag time (h). The cumulative amount  of caffeine permeated across the skin (corrected for acceptor phase replacement) was plotted against time (h), and the steady-state flux JSS (μg cm−2 h−1) was calculated from the linear region of the slope of the plot. The permeability coefficients (Kp) were calculated according to the equation: Kp = Jss/Cd, where Cd is the initial drug concentration. The lag time was interpreted as the intercept with the x-axis. The influence of PDM and EO on the caffeine permeation profile (penetration-enhancing activities) was expressed as the enhancement index (EI), i.e., the ratio of the Kp value with an enhancer to that obtained without an enhancer.The data obtained were analyzed for a cumulative amount of drug permeated . Approximately 24 h before the test, fur was shaved from the dorsal area, and caffeine and caffeine plus 1% EO or PDM hydrogels were gently applied over the shaved area. A separate, untreated site was used as a control. The treatment was performed once a day for 21 days. Signs of edema or erythema at the application site after 4, 24, and 72 h and after 9 and 14 days post-treatment were registered and scored from 0 (no irritation) to 4 (severe irritation).p was less than 0.05. Data are presented as the means ± SEMS, and the number of replicates (n) is given in the pertinent figures.Using the statistical program GraphPad Prism 8.1 , one-way analysis of variance (ANOVA) was used to analyze each experiment, along with post hoc comparisons (Tukey Dunnett’s test). Comparisons were made between the caffeine gel formulations without and with EO or PMD. A result was considered significant when L. origanoides (EO19) followed by sesquiterpene-rich T. diffusa (EO2) enhanced caffeine delivery via the transdermal route, still higher than eugenol, carvacrol, and limonene, which are recognized and confirmed PE terpenoids. No skin irritation was observed when 1% EOs and PDM hydrogels were used for at least 15 days. Many publications revised in this paper provide substantial evidence that some EO and their terpene constituents are interesting PEs for TDD of low-MW  and hydrophilic or lipophilic (LogP from −3.03 to 4.5) drugs. However, important contributions of this work lie in the fact that our enhancer formulations were still effective at low EO or terpene concentrations, were non-irritants, and were potentially able to be used in humans in the case of prolonged treatments (21 days). In addition, L. origanoides and T. diffusa plants are now cultivated in some Colombian regions to produce EOs at industrial scale for their analysis, applications, and commercial use.The findings demonstrate that thymol-rich


Accumulated evidence indicates that geniposide exhibits neuroprotective effects in ischemic stroke. However, the potential targets of geniposide remain unclear.We explore the potential targets of geniposide in ischemic stroke.in vivo and in vitro.Adult male C57BL/6 mice were subjected to the middle cerebral artery occlusion (MCAO) model. Mice were randomly divided into five groups: Sham, MCAO, and geniposide-treated (i.p. twice daily for 3 days before MCAO) at doses of 25, 75, or 150 mg/kg. We first examined the neuroprotective effects of geniposide. Then, we further explored via biological information analysis and verified the underlying mechanism P < 0.05) improved neurological deficits, brain edema (79.00 ± 0.57% vs 82.28 ± 0.53%), and infarct volume (45.10 ± 0.24% vs 54.73 ± 2.87%) at 24 h after MCAO. Biological information analysis showed that the protective effect was closely related to the inflammatory response. Geniposide suppressed interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) expression in the brain homogenate, as measured by enzyme-linked immunosorbent assay (ELISA). Geniposide upregulated A20 and downregulated TNF receptor-associated factor-6 and nuclear factor kappa-B phosphorylation in the MCAO model and lipopolysaccharide-treated BV2 cells at 100 μM.In the current study, geniposide had no toxicity at concentrations of up to 150 mg/kg. Compared with the MCAO group, the 150 mg/kg group of geniposide significantly (in vivo and in vitro experiments, which may provide a potential direction for the application of geniposide in the treatment of ischemic stroke.Geniposide exhibited a neuroprotective effect via attenuating inflammatory response, as indicated by biological information analysis,  BCAbicinchoninic acidBPbiological processCCcellular componentDAMPsdamage-associated molecular patternsECAexternal carotid arteryELISAenzyme-linked immunosorbent assayGDgeniposideGOgene ontologyHRPhorseradish peroxidase;IRAKIL-1 receptor-associated kinaseIL-6interleukin-6iNOSinducible nitric oxide synthaseLPSlipopolysaccharideMCAOmiddle cerebral artery occlusionMFmolecular functionNF-κBnuclear factor kappa-BPPRspattern recognition receptorsPVDFpolyvinylidene difluoriderCBFregional cerebral blood flowRIPAradioimmunoprecipitation assay bufferTLRstoll-like receptorsTRAF6TNF receptor-associated factor-61S,4aS,7aS)-1-(β-d-glucopyranosyloxy)-7-(hydroxylmethyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate; C17H24O10) is an iridoid glucoside extracted from Gardenia jasminoides Ellis (Rubiaceae) fruits, a flowering plant [Geniposide  and the TNF receptor pathway ,13. As aIn the current study, we explore whether geniposide exhibited neuroprotective effect during cerebral ischemia and whether the neuroprotective effect was mediated through the A20/NF-κB signal pathway.22.1Animals were housed in a humidity-controlled room on a 12 h light/dark cycle at 22°C with free access to food and water. Adult male C57BL/6 mice (8–10 weeks) were used for the experiments. Animals were assigned randomly using a random number table. All assessments were conducted by investigators who were blinded to the experimental group assignment.A total of 150 adult male C57BL/6 mice (weighing 25–30 g) were used in our current study; 14 (9.3%) mice died before the completion of the experiment and were excluded from the study. Post-mortem examinations did not reveal the occurrence of intracerebral or subarachnoid hemorrhage in any of these animals, and no significant differences were found in the number of deaths in each group.Ethical statement: All experimental procedures were approved by the committee of experimental animals of Hebei Medical University, Shijiazhuang, China (2020-AE006), and conducted in accordance with the National Institutes of Health guidelines. 2.2Geniposide (purity >98% tested by HPLC) was purchased from Chengdu Derick Biotechnology Co. Ltd. . Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. . Antibodies against P-p65 , p65 , A20 , and GAPDH  were purchased from Cell Signaling Technology . TRAF6  was purchased from Thermo Fisher Scientific. Tumor necrosis factor-α , IL-6 , interleukin-1β , and iNOS ELISA kits were purchased from BioLegend .2.3Geniposide  with a purity of more than 98% was dissolved in saline. All the mice were randomly divided into five groups as follows: sham group, middle cerebral artery occlusion (MCAO) group, GD25 (25 mg/kg geniposide) group, GD75 (75 mg/kg geniposide) group, and GD150 (150 mg/kg geniposide) group. Sham and MCAO groups were treated with 0.9% normal saline only through intraperitoneal injection. Geniposide was administered through intraperitoneal injection with doses of 25, 75, or 150 mg/kg twice daily for 3 days before surgery.2.4Mice were anesthetized with 1% pentobarbital . Permanent focal cerebral ischemia was induced by right MCAO using an intraluminal filament occlusion model as previously reported . First, 2.5n = 10 per group). The neurological deficits were scored using a modified scoring system reported previously [The neurological deficits of mice were conducted before the mice were euthanized at 24 h after MCAO by an examiner blinded to the groups  were anesthetized by pentobarbital, and brains were rapidly removed on the ice. Brains were sliced into five 2-mm-thick coronal sections and then stained with 2% 2,3,5-triphenyltetrazolium chloride  for 10 min at 37°C. The infarct volume was calculated as follows:  [At 24 h after MCAO, mice ( volume) .2.7n = 6 per group). The two hemispheres of mice were packaged with tinfoil, respectively, to obtain wet weights on an electronic balance, and dry weights were then obtained after placing them into the oven for 24 h at 100°C. The brain water (BW) content was then calculated as follows:Brain edema was evaluated by the standard wet/dry method  at 24 h BW = (wet weight − dry weight)/wet weight × 100%.2.8g for 15 min at 4°C to collect the supernatant. The procedure was performed following the manufacturer’s instructions. Briefly, 50 mM carbonate coating buffer (pH 9.6) was used to dissolve the antigen (10–20 μg/mL), which was then added into a 96-well enzyme label plate (100 μL/well) for overnight incubation at 4°C. Then, 150 μL of 1% bovine serum albumin was added to each well for 1 h of blocking at 37°C, and 100 μL of serum was added into each well to incubate for 2 h at 37°C. Subsequently, the serum was incubated with 100 μL of diluted horseradish peroxidase (HRP)-labeled secondary antibodies at 37°C for 1 h. Finally, the absorbance value A405 was read on a microplate reader.TNF-α, IL-6, IL-1β, and iNOS levels in the brain homogenate of the ischemic hemisphere were determined using a commercial ELISA kit  at 24 h after MCAO. The brain homogenate was then centrifuged at 1,000×2.9Proteins from the mice brains and total proteins from the treated cells were extracted with the radioimmunoprecipitation assay buffer (RIPA) and were determined using the bicinchoninic acid (BCA) protein assay kit, as previously reported . Equal a2.102/95% air). To test the effect of geniposide on cell viability, the cells were divided into a control group, 5 μM group (5 μM geniposide), 10 μM group (10 μM geniposide), 20 μM group (20 μM geniposide), and 100 μM group (100 μM geniposide). To explore the effect of geniposide on inflammation, LPS was administered at a concentration of 200 ng/mL. Cells were classified into different groups: control group, LPS group, and LPS + GD group (100 μM geniposide).BV2 cells, a microglial cell line, were cultured in Dulbecco's modified Eagle medium (DMEM) solution , supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin , in an incubator under normoxic conditions  is an online analysis software, which can predict the target of small molecules according to the principle of molecular similarity. We utilized this tool to predict the targets of geniposide. A protein–protein interaction (PPI) network was constructed through the STRING  database, which is a database of known and predicted PPIs, including direct  and indirect  associations.SwissTargetPrediction (2.13the David 6.8 (https://david.ncifcrf.gov/) database, which provides a comprehensive set of functional annotation tools to elaborate the biological function of the long list of genes. Briefly, to start DAVID, first click on “Functional Annotation” under “Shortcut to David tools” at the left of the home page. Then, we upload the file containing the targets of geniposide, which were predicted through SwissTargetPrediction. After the file was uploaded, we select “Functional Annotation Clustering” in the center of the page. The GO terms obtained in the drawing are arranged in descending order according to the −log10  of enrichment, and we used the first 30 results.GO analysis consists of three parts: molecular function, biological process, and cellular component. Enrichment analysis was performed via 2.14t-test for two-group comparisons. For neurological deficits, Mann–Whitney U test was used for comparisons between the two groups. Fisher’s exact test was applied for the GO analyses. P < 0.05 was considered statistically significant.All data were expressed as mean ± SEM. For statistics normally distributed, comparisons were assessed by one-way ANOVA followed by Student–Newman–Keuls tests and least significant difference (LSD) tests for multi-group comparisons and two-tailed Student’s 33.1The flow chart of our experiment is shown in P < 0.05, respectively) when compared with the MCAO group. However, no obvious difference was observed among the groups of MCAO and 25 mg/kg GD group.Brain edema was measured at 24 h after MCAO using wet/dry methods, as shown in P < 0.05, respectively) when compared with the MCAO group. No obvious difference in the cerebral infarction volume was observed in the MCAO and 25 mg/kg GD group.Infarct volume was evaluated at 24 h after MCAO by staining, through which viable tissue is stained red and ischemic tissue is stained pale, as shown in These results indicated that geniposide exerted a neuroprotective effect after cerebral ischemic injury. Based on the above-mentioned results, we selected 150 mg/kg GD for the following study.3.2To predict the potential targets of geniposide, we queried geniposide targets using the SwissTargetPrediction tool. One hundred geniposide targets were predicted with SwissTargetPrediction . To construct an interaction network, we studied the PPI relationship via the STRING database. This network consisted of the above 100 terms, including 100 nodes and 400 edges . As show3.3To investigate the biological function and potential mechanism of geniposide, GO analyses were performed via the DAVID database. Biological process analysis was mainly enriched in terms of inflammatory response and negative regulation of apoptotic progress. Molecular function analysis was primarily enriched in terms of protein binding . These f3.4To confirm the accuracy of target prediction and GO analysis, we thus investigated the expression of pro-inflammatory cytokines in the brain at 24 h after cerebral ischemia. The results showed that the expression of TNF-α, IL-6, IL-1β, and iNOS was upregulated after MCAO . Importa3.5We further explored the expression of A20, TRAF6, and the phosphorylation of NF-κB, which was the upstream signal of IL-6 and iNOS, in the penumbra of the brain tissue. As shown in As IL-6 and iNOS were mainly produced by activated microglia, we used microglia cell lines, BV2 cells, to examine the inhibitory effect of geniposide on microglia activation and its underlying mechanisms.To evaluate whether geniposide affected the cell viability of BV2 cells, the cell viability was assessed by CCK8 assay. As shown in LPS (200 ng/mL) was then used to activate microglia. As shown in 4In the current study, we first investigated the neuroprotective role of geniposide during cerebral ischemia in mice and then explored the potential targets of geniposide using bioinformatics tools, including SwissTargetPrediction and analyses of the GO pathway and PPI network. With the bioinformatics tools, we identified that the protective effect of geniposide may be mediated through its anti-inflammatory properties. We then verified the anti-inflammatory effect of geniposide and revealed that A20 may serve as a novel target in the treatment of cerebral ischemia.G. jasminoides fruits, such as the Bazheng mixture, Longdan Xiegan pills, Qingkailing soft capsules, Niuhuang Shangqing soft capsules, and Zhizi Jinhua pills . Multiple lines of pharmacological evidence have demonstrated the various biological properties of geniposide, such as anti-inflammatory [in vivo; 25, 50, and 100 µg/mL in vitro) has been demonstrated to alleviate inflammatory response by the inhibition of TLR4. Moreover, evidence has shown that geniposide may serve as a potential drug to prevent the early progression of AD [Geniposide is found in 40 species belonging to various families, especially Rubiaceae . In the ammatory , antioxiammatory , antidiaammatory , and neuammatory . In a LPon of AD . Geniposon of AD . CurrentThrough the SwissTargetPrediction database, we verified 100 geniposide targets, which were enriched in the pathway of inflammatory and cell apoptosis as indicated by GO analysis. Accumulated evidence has demonstrated that inflammation plays a crucial role after cerebral ischemia . After cMicroglia, identified as the macrophage of the central nervous system, play important roles in neuroinflammation after cerebral ischemia . After cImportantly, we demonstrated that geniposide could upregulate the expression of A20 after cerebral ischemia, which was not found as a geniposide target via SwissTargetPrediction but was predicted with the highest score in GEO datasets of cerebral ischemia, suggesting that A20 was a potential geniposide target. A20, a zinc finger protein, functions as a potent negative regulator of the inflammatory response and feedback inhibitor of NF-κB. Mounting evidence has demonstrated that A20 could regulate the activity of TRAF6 by direct K63-linked deubiquitination and by K48-linked ubiquitination via the ubiquitin–proteasome system. These results suggested that geniposide inhibited the expression of TRAF6 and the phosphorylation of NF-κB, at least partly, by increasing the expression of A20 .5Our current study suggested that upregulation of A20 was involved in the neuroprotective and anti-inflammatory effect of geniposide during cerebral ischemia. The neuroprotective effect of geniposide was, at least partly, through the A20/NF-κB pathway.


The Sunahara method was used to extract total rifamycins, and rifampin urine excretion was measured by spectrophotometry. An analysis of 58 eligible participants, including 15 (26%) with type 2 diabetes mellitus, demonstrated that urine spectrophotometry accurately identified subtarget rifampin AUC0–24 at 0–4, 0–8, and 0–24 h. The area under the receiver operator characteristic curve (AUC ROC) values were 0.80 (95% CI 0.67–0.90), 0.84 (95% CI 0.72–0.94), and 0.83 (95% CI 0.72–0.93), respectively. These values were comparable to the AUC ROC of 2 h serum concentrations commonly used for therapeutic monitoring . Diabetes status did not significantly affect the AUC ROCs for urine in predicting subtarget rifampin serum exposure (P = 0.67–0.92). Spectrophotometric measurement of urine rifampin excretion within the first 4 or 8 h after dosing is a simple and cost-effective test that accurately predicts rifampin underexposure. This test provides critical information for optimizing tuberculosis treatment outcomes by facilitating appropriate dose adjustments.Variable pharmacokinetics of rifampin in tuberculosis (TB) treatment can lead to poor outcomes. Urine spectrophotometry is simpler and more accessible than recommended serum-based drug monitoring, but its optimal efficacy in predicting serum rifampin underexposure in adults with TB remains uncertain. Adult TB patients in New Jersey and Virginia receiving rifampin-containing regimens were enrolled. Serum and urine samples were collected over 24 h. Rifampin serum concentrations were measured using validated liquid chromatography–tandem mass spectrometry, and total exposure (area under the concentration–time curve) over 24 h (AUC Rifampin is a key sterilizing drug in standard tuberculosis (TB) regimens that enables cure of most drug-susceptible TB within 6 mo. However, there is extensive pharmacokinetic (PK) variability in absorption and metabolism of anti-TB drugs including rifampin, with low drug concentrations associated with poor treatment outcomes including treatment failure, relapse, and acquired resistance , 2. To g0–24 exposures  , 6. In pThe study enrolled adults undergoing treatment for active TB with a first-line rifampin-containing regimen during an intensive  or continuation (rifampin+isoniazid) phase of anti-TB therapy at recommended doses . Partici0–24), was determined by noncompartmental analysis using Phoenix WinNonlin version 8.3  , then at 1, 2, 4, 6, and 8 h post-dose . Blood sey, USA) , 11.Urine was collected at baseline (predose) and 0–4, 4–8, and 8–24 h (post-dose). Separate graded receptacles were used for each urine collection interval. At 0–4 and 4–8 h, aliquots were drawn from the collection receptacle, and the total urine volume at each timepoint was noted. Three different urine collection periods were examined: 0–4, 0–8, and 0–24 h. Proportional volumes of urine collected at 0–4, 4–8, and 8–24 h post-dose were pooled to determine concentrations at 0–8 and 0–24 h to reflect potential collection times in a clinical setting.The Sunahara method was followed to extract total rifamycins, including rifampin and its metabolites, in urine . For eve0–24 for each participant that completed the study visit. For our primary analysis, we evaluated receiver operating characteristic (ROC) curves for the urine spectrophotometric assay corresponding to an AUC0–24 target of 35.4 mg·h/L, representing a contemporary consensus drug exposure target , with ROC analysis performed using the pROC package  and creatinine clearance. Diabetes status was defined in this study as a measured HbA1c ≥ 6.5% or an established diagnosis of diabetes on treatment, regardless of HbA1c. Data were analyzed in R software .Serum rifampin concentrations for the 58 individuals are shown in 2r = 1) between rifampin concentration from 31.2 to 1,000 mg/L at the University of Virginia laboratory and between 7.8 and 1,000 mg/L at the Rutgers laboratory, due to different instrument detection limits. Urine from five participants was evaluated for assay reproducibility at both institutions, and the calculated rifampin concentration variation was under 10%. The distribution of urine excretion amounts by participant is shown in 0–24 values of 2.4, 21.4, and 31.9 mg·h/L. There was no association between urine excretion and number of days on rifampin treatment (0 > R > −0.029).The calibration curve generated to quantify extracted rifampin from urine via the Sunahara method demonstrated a linear relationship , 0–8 h 0.84 , and 0–24 h 0.83  .For predicting those participants with serum rifampin below the minimum target AUC < 0.01) . There w0–24 below the target of 13 mg·h/L. Urine excretion maintained an AUC ROC >0.8 in predicting serum AUC0–24 below the target of 13 mg·h/L, although serum 2 h concentration tended to be more predictive at this threshold with an AUC ROC of 0.94 .Only four participants had an AUCP < 0.01), 0.86 , and 0.85 , respectively . Among the remaining 55 individuals with CrCl >60 mL/min, the AUC ROCs of urine 0–4, 0–8, and 0–24 were 0.81 . Urinalysis profiles associated with diabetes were considered with regard to potential impact on rifampin excretion and measurement. Among the 14 participants with diabetes versus 39 without diabetes with available urinalysis data, 35.7% (5/14) vs 23.1% (9/39) had trace to 2+ proteinuria, 57.1% (8/14) vs 0% (0/39) had glucosuria, and average pH was 5.9 and 5.8, respectively.Diabetes and sex had no significant impact on the AUC for ROC curve of urine at any interval or serum 2 h concentration for predicting AUC0–24 across each urine time interval and serum 2 h when setting the sensitivity threshold to 80% or 90% and at the maximum Youden index. At minimum sensitivity thresholds of 80% or 90%, specificity of urine spectrophotometry including at 0–4 and 0–8 intervals met or exceeded that of serum 2 h values. At the maximum Youden index, sensitivity exceeded 86% for urine at 0–4 and 0–8 h.Given the wide therapeutic window and tolerability of rifamycins , 18, we Spectrophotometric measurement of urine rifampin excretion over 24 h, even in the first 4 and 8 h post-dose, accurately predicted relevant subtarget rifampin serum exposures with an AUC ROC of 0.8 or greater and was no different than that of a 2 h serum concentration for predicting a clinically relevant target of total serum exposure. In contrast to collection and measurement of serum concentrations, urine spectrophotometry is a low-cost method without the need for specialized expertise or equipment and imminent potential to be developed for point-of-care or near-point-of-care use.0–24 values averaging 308 mg·h/L have been tolerated with mild, grade 1–2 hepatotoxicity   , and rese range) , 21.One limitation of this study is the modest sample size to perform further subgroup analyses. Despite this limitation, we observed that urine spectrophotometry performed well in both patients with and without diabetes despite observed differences that were expected in those groups with regard to urinalysis profiles. We have recently demonstrated similar predictive performance for serum rifampin exposures at different urine collection intervals among a more malnourished cohort of children treated for TB in Tanzania . Future In conclusion, urine spectrophotometry can accurately diagnose rifampin serum pharmacokinetic underexposure. The noninvasive and low-cost characteristics of urine spectrophotometry make it a preferential option for use in TB endemic or programmatic settings that previously have not benefited from personalized dosing. Urine spectrophotometry could be scaled up to ensure adequate minimal drug exposure in all patients by continued used of fixed-dose combinations plus single additive rifampin tablets, thereby ameliorating one of the few correctable determinants of TB treatment outcomes.