Verticillium dahliae grown in three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we observe local quantitative differences in H3K27me3 ChIP-seq signals that are associated with a subset of differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta-induced genes may require chromatin remodeling to achieve expression. Overall, our results demonstrate that some loci display H3K27me3 dynamics associated with concomitant transcriptional variation, but many differentially expressed genes are associated with stable H3K27me3 domains. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it does not appear that transcriptional activation requires the global erasure of H3K27me3. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions.Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far, no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics govern differential transcription. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing data from the plant-pathogenic fungus  Saccharomyces cerevisiae senses cold stress by increased membrane rigidity, which leads to the transcription of genes that, among others, encode cell damage-preventing proteins  regions that are variable between strains and enriched for genes that are in planta induced , chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase accessible chromatin followed by sequencing (ATAC-seq) data for V. dahliae cultured in various axenic growth media to understand if transcriptional dynamics require concomitant changes in the H3K27me3 modification status.The filamentous fungus  disease . Genomic induced , 40. The induced –44. Recenduction –24, we nnfection , which infection . Neverthnfection , 17, we V. dahliae, we mapped the occurrence of the heterochromatic histone marks H3K9me3 and H3K27me3, the euchromatic histone marks dimethylation of lysine 4 on histone H3 (H3K4me2) and acetylation of lysine 27 on histone H3 (H3K27ac), and chromatin accessibility determined by ATAC-seq  displayed low H3K4me2 and H3K27ac coverage upstream of the transcription start site (TSS), followed by a steep increase of coverage over the start of the gene that decreased over the gene body and increased again at the transcription end site (TES)  . The strte (TES) . The lowte (TES) ; Fig. S2 regions ; Fig. S2 regions , 48, 49. regions ; Fig. S2 regions , 27, 32.10.1128/mbio.03566-21.1FIG S1FIG S1, PDF file, 0.3 MB.Distribution of chromatin features over all chromosomes. Download Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03566-21.2FIG S2FIG S2, PDF file, 0.2 MB.Gene expression correlates with histone modification presence and chromatin accessibility. Download Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the V. dahliae in PDB. These 3,186 genes were separated into five expression quintiles as described above. Interestingly, we found that genes in H3K27me3 domains with higher expression values (quintiles 1 and 2) had higher H3K4me2 values over the gene body, more accessible promoters, and lower H3K27me3 values  from triplicate-grown 3 values . This suV. dahliae, we deleted the histone methyltransferase component of polycomb repressive complex 2 (PRC2), termed Set7 (ΔSet7), leading to the loss of H3K27me3  , 625 (74.5%) are located in H3K27me3 domains, which is significantly higher than expected given that only 27.9% of genes are located in H3K27me3 domains (P < 0.00001 by Fisher’s exact test). In contrast, we find that 211 (27.6%) of 765 repressed genes in the ΔSet7 mutant are in H3K27me3 domains   (in vitro.To further characterize the influence of H3K27me3 on gene expression in H3K27me3  and S4. ype (WT) . As H3K2ct test)  and C. A t test) . These f10.1128/mbio.03566-21.3FIG S3V. dahliae ΔSet7 deletion mutant. Download FIG S3, PDF file, 0.06 MB.Western blot showing the loss of H3K27me3 in the Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03566-21.4FIG S4V. dahliae ΔSet7 mutant. Download FIG S4, PDF file, 0.1 MB.ChIP sequencing shows the loss of H3K27me3 in the Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03566-21.5FIG S5V. dahliae are more strongly transcriptionally induced in the ΔSet7 mutant than non-H3K27me3-associated genes. Download FIG S5, PDF file, 0.06 MB.Genes associated with H3K27me3 in wild-type Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the V. dahliae growth conditions that lead to differential expression. Here, the observed changes in H3K27me3 domains should be associated with transcriptional differences of the underlying genes. An alternative hypothesis is that the H3K27me3 domain status does not change in accordance with transcriptional activity, and the repressive effects of H3K27me3 are released through alternative means. To test this hypothesis, we performed triplicates of H3K27me3 ChIP-seq on V. dahliae cultivated for 6 days in Murashige-Skoog medium (MS) and Czapek-Dox medium (CZA), in addition to the previously generated ChIP-seq data in PDB. Based on the correlation between replicates , but they are more highly expressed in CZA than in both PDB (P = 2e−4 by a one-sample t test) and MS (P = 5e−8 by a one-sample t test) (P = 0.92 by a one-sample t test); when the genes are associated with H3K27me3, they are more highly expressed in MS than in PDB (P = 4e−3 by a one-sample t test) but not more highly expressed in MS than in CZA (P = 0.99 by a one-sample t test). For genes present in shared H3K27me3 domains between MS and CZA growth (223 genes), we did not observe statistically significant transcriptional differences between the growth conditions where the genes lacked H3K27me3 domains  . Similar domains  and C. A domains . The gen domains . Overall10.1128/mbio.03566-21.6FIG S6FIG S6, PDF file, 0.10 MB.Correlation between H3K27me3 ChIP samples. Download Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03566-21.7FIG S7FIG S7, PDF file, 0.1 MB.Differentially expressed genes are enriched in H3K27me3 domains during cultivation in nonpermissive growth medium. Download Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 fold change for H3K27me3 coverage, indicating that these genes have lower H3K27me3 signals in PDB than in MS   . Overalld change , and F. V. dahliae is host colonization and the contrast for the genes differentially expressed in planta compared with axenic culture. We have thus far been unable to perform ChIP on V. dahliae during host colonization due to a low pathogen-to-plant biomass, and therefore, we cannot compare H3K27me3 levels between these conditions. However, we can identify the genes that are significantly induced during infection compared to in vitro growth media and assess their chromatin profiles in these media to assess if in planta-induced genes appear heterochromatic during in vitro growth. Genes were grouped based on differential expression between PDB and in planta growth, and chromatin profiles showed that genes that are more highly expressed in planta have significantly more H3K27me3 during PDB growth than genes that are more highly expressed in PDB , 15.5% had a secretion signal, and 4.3% were predicted effectors. This is compared to all in planta-induced genes relative to PDB , where 12.3% had a secretion signal and 2.7% were predicted effectors. These results were similar between the other two media, and we conclude that in planta-induced genes in H3K27me3 domains have a slightly higher fraction of genes that are potentially involved in infection than in planta-induced genes not in H3K27me3 domains. Collectively, these results indicate that genes that are more highly expressed in planta have a heterochromatic profile  when analyzed in axenic culture. The chromatin profile of these genes during in planta growth will need to be directly assessed in the future.The presented analyses compared the directionality of H3K27me3 and transcriptional changes between axenic growth media to address if H3K27me3 dynamics are associated with transcriptional dynamics. Another interesting growth condition for d in PDB . These iB growth . The ass and CZA  and C. Fee media . We foun domains . We obsein vitro growth conditions are located in H3K27me3 domains. This is interesting because the mark contributes to transcriptional repression, and it is relatively sparse in the genome, occupying approximately 33% of the genes. However, 35% to 70% of differentially expressed genes identified between growth conditions reside within H3K27me3 domains. It is not clear what mechanism might drive this association.Chemical histone modifications play an essential role in transcriptional regulation, but a number of mechanistic questions remain for their role in differential transcription in filamentous fungi. In this study, we show that many genes that are differentially transcribed between V. dahliae is similar to what has been reported for the ascomycete Podospora anserina   , 32, 50.. oryzae , 32, 50.in vitro growth conditions indicates that this can account for some of the observed transcriptional differences. For example, some genes have a lower H3K27me3 ChIP signal upon growth in transcriptionally permissive medium than upon growth in transcriptionally repressive medium. We interpret these results as evidence for local, rather than global, changes in H3K27me3 dynamics contributing to transcriptional regulation for the underlying genes. H3K27me3-associated genes that are differentially expressed while the presence of the histone mark remains stable may be transcriptionally regulated through the activity of H3K27me3 readers. For instance, the Fusarium graminearum histone reader BP1, which is orthologous to N. crassa EPR-1, specifically binds to H3K27me3 and corepresses gene transcription  showed that some, but not all, analyzed regions displayed differential H3K27me3 levels consistent with increased transcription between in planta and in vitro conditions  may yield a different picture of the global H3K27me3 distribution. Additionally, our experiments focused on steady-state growth on different media as these provide a clear and reproducible transcriptional difference. It is possible that H3K27me3 dynamics occur rapidly in response to environmental changes, cues, or developmental stages that we did not capture.We note that an individual histone does not permit a quantitative trimethylation status, as an individual H3K27 is either trimethylated or not. At the nucleosome level, there can be no, one, or two H3K27 tails with a trimethyl modification. At the cell population level, there can be considerable quantitative differences because of the heterogeneity for histone modification status between individual cells. Cell variability may be the source of the quantitative differences observed in our experiments, arising from variation in both the percentage of cells with H3K27me3 and the number of tails of a nucleosome with H3K27me3. While we cannot determine this based on our present data, our results show that some genes that are differentially expressed between growth conditions are associated with quantitative differences in H3K27me3 levels, providing evidence that chemical histone modification dynamics can be involved in a transcriptional regulatory mechanism in nditions . We haveN. crassa have shown that genetic perturbation leading to the genome-wide loss of H3K9me3 causes a redistribution of H3K27me3 to previously H3K9me3-marked sites, but interestingly, this redistribution does not result in transcriptional silencing  (4 spores per mL into liquid growth media. Media used in this study are potato dextrose broth (PDB) , half-strength Murashige-Skoog medium plus vitamins (MS)  supplemented with 3% sucrose, and Czapek-Dox medium (CZA) . Liquid cultures were grown for 6 days in the dark at 22°C at 140 rpm. The mycelium was collected by straining cultures through Miracloth (22 μm)  and pressing to remove liquid, after which the mycelium was flash-frozen in liquid nitrogen and ground to a powder with a mortar and pestle. If required, samples were stored at −20°C prior to nucleic acid extraction. All analyses were performed based on triplicate cultures that were processed individually. 143773)  was cultRNA was isolated from ground mycelium using TRIzol  according to the manufacturer’s guidelines. Contaminating DNA was removed using the Turbo DNA-free kit , and RNA integrity was assessed by gel electrophoresis and quantified using a Nanodrop spectrophotometer . Libraries were prepared and single-end 50-bp sequenced on the DNBseq platform at BGI .V. dahliae strain JR2  was constructed as previously described (Agrobacterium tumefaciens strain AGL1 and used for V. dahliae conidiospore transformation as described previously (V. dahliae transformants that appeared on hygromycin B were transferred to fresh PDA supplemented with hygromycin B after 5 days. Putative transformants were screened using PCR to verify the deletion of the target gene sequence and the integration of the selection marker at the designated locus using primers listed in Set7 deletion mutant to that in wild-type V. dahliae were performed as described above.The escribed . Brieflyescribed  using Useviously . V. dahl10.1128/mbio.03566-21.9TABLE S1Set7 coding sequence in V. dahliae. Download Table S1, PDF file, 0.06 MB.Primers used to delete and analyze the Copyright © 2022 Kramer et al.2022Kramer et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Lentilactobacillus buchneri isolated from Korean fermented plant foods produces β-glucosidase, which can hydrolyze ginsenoside Rb1 from Panax ginseng to yield ginsenoside Rd. The aim of this study was to determine the mechanisms underlying the extracellular β-glucosidase activity obtained from Lentilactobacillus buchneri URN103L. Among the 17 types of lactic acid bacteria showing positive β-glucosidase activity in the esculin iron agar test, only URN103L was found to exhibit high hydrolytic activity on ginsenoside Rb1. The strain showed 99% homology with Lentilactobacillus buchneri NRRLB 30929, whereby it was named Lentilactobacillus buchneri URN103L. Supernatants of selected cultures with β-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40 °C, pH 5.0. Furthermore, the β-glucosidase activity of this strain showed a distinct ability to hydrolyze major ginsenoside Rb1 into minor ginsenosides Rd and Rg3. Lentilactobacillus buchneri URN103L showed higher leucine arylamidase, valine arylamidase, α-galactosidass, β–galactosidase, and β-glucosidase activities than any other strain. We conclude that β-glucosidase from Lentilactobacillus buchneri URN103L can effectively hydrolyze ginsenoside Rb1 into Rd and Rg3. The converted ginsenoside can be used in functional foods, yogurts, beverage products, cosmetics, and other health products. Panax ginseng C.A. Mey) contains approximately 200 substances, including ginsenosides, polysaccharides, polyacetylenes, peptides, and amino acids. To date, 100 ginsenosides have been isolated, with Rb1, Rb2, Rc, Re, and Rg1 constituting more than 80% of all ginsenosides reported [Korean ginseng , Re (12.95%), Rc (11.12%), Rd (5.87%), Rb2 (8.32%), Rb1 (15.91%)) in a 1:1 (v/v) ratio. The mixture of supernatant and standard saponin was incubated at 35 °C for 7 days at 190 rpm in a shaking incubator . The hydrolysis of saponin was monitored by thin-layer chromatography (TLC) analysis.Lactobacillus strains were isolated from eight types of traditional Korean fermented plant foods randomly collected from the Daejeon area. Isolation of LAB with β-glucosidase activity from fermented plants was performed according to the method in . The cul16S rDNA sequencing of strain URN103L was performed according to the method in . The priEnzyme activity patterns of the selected strains were investigated using the API ZYM Kit for the research of enzymatic activity , following manufacturer instructions .L. buchneri URN103L was inoculated in MRS broth at 37 °C for 24 h, after which the supernatant was collected by centrifugation at 7000× g for 15 min at 4 °C. The crude enzyme extract of the selected cultures was tested for the hydrolysis of major ginsenoside Rb1  by β-glucosidase. The reaction mixture was prepared in a 1:1 (v/v) ratio of supernatant and 0.2 mg mL−1 of ginsenoside Rb1 (5 mM of sodium phosphate buffer) and filtered through a 0.2 μm syringe filter . The reaction mixture was incubated at 35 °C for 7 days. β-glucosidase activity of the supernatant was conducted according to the method in [L. buchneri URN103L was determined at pH 7.0 at 30, 35, and 40 °C, over 7 days. Then, samples were extracted with butanol  prior to their concentration using a freeze-drying machine . Finally, the ginsenoside concentration was adjusted to 1 mg mL−1 for TLC analysis.The optimum temperature for the hydrolysis of ginsenoside Rb1 by the crude enzyme extract of L. buchneri URN103L were analyzed by TLC and HPLC, according to the method in [Fermentation characteristics of ginsenosides fermented by the strain ethod in .L. buchneri URN103L was inoculated in MRS broth (pH 7.0), and the crude enzyme extract was collected by centrifugation at 7000× g for 15 min at 4 °C. The reaction mixture in ratio 1:1 (v/v) of crude enzyme extract (pH 5.0) and 0.2 mg mL−1 of ginsenoside Rb1  was filtered through a 0.2 μm syringe filter and incubated at 35 °C for 3, 7, 10, and 14 days. Samples were concentrated using a freeze-drying machine . Finally, the ginsenoside concentration was adjusted to 1 mg mL−1 for TLC and HPLC.The hydrolysis of ginsenoside Rb1 was performed under the optimum conditions determined for β-glucosidase activity as described above. L. buchneri URN103L were analyzed by TLC and HPLC, according to the method in [Ginseng roots were sliced using a mixer for 5 min, diluted to 20%, and sterilized at 75 °C for 10 min. The resultant 20% ginseng root solution was incubated with 3% strain URN103L at 35 °C for 14 days. After 3, 7, and 14 days, viable cell counts were determined using BCP agar incubated at 37 °C for 48 h. Fermentation characteristics of ginsenosides fermented by the strain ethod in .p < 0.05). Data are presented as mean ± standard error.All experiments were repeated in triplicates. One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were conducted using SAS  to measure significant differences  production ability, and the GABA produced by the isolate was found to have a neuroprotective function and to protect MPP+-stressed SH-SY5Y cells more effectively than other LAB [L. buchneri KU200793 can reportedly be used as a probiotic starter in functional foods to prevent Parkinson’s disease [L. buchneri P2 isolated from pickled juice has shown probiotic properties, such as cholesterol reduction, bile tolerance, and antimicrobial activity [L. buchneri URN103L with β-glucosidase activity that can hydrolyze ginsenoside from ginseng obtained from fermented plants.Thirty-seven types of pure culture of bacteria from fermented plants were selected in this study. The characteristics of ginsenoside hydrolysis and the utilization of esculin agar for the β-glucosidase production ability at 37 °C, over 72 h, were carefully investigated. Seventeen strains  among the fermented plant isolates showed an esculin-positive reaction. Furthermore, among these esculin-positive colonies, eight  showed hydrolysis of ginsenoside Rb1 and were analyzed for 16S rDNA sequencing. Isolated single colonies of strain 103L in BCP and esculin agar are shown in operties . Indeed, disease . Additioactivity . In thisAmong the selected colonies, strain URN103L actively metabolized major ginsenoside Rb1 to minor ginsenoside Rd. However, this strain did not metabolize ginsenoside Rb1 to ginsenosides Rb2 and Re. Finally, TLC analysis  showed tL. rhamnosus GG and L. buchneri KU20073 showed the highest level of β-glucosidase, α-glucosidase, and β-galactosidase activities, which are important enzymes for the hydrolysis of glycosidic linkages [L. buchneri KU20073 produces leucine arylamidase and valine arylamidase with the highest activity, similar to isolated strain URN103L [L. casei subsp. rhamnosus [Leuconostos mesenteroides [Lactobacillus plantarum [API ZYM is a simple, rapid system for the detection of bacterial enzymes that allows the detection of enzyme activity on 19 substrates within 4 h [linkages . Strain  URN103L . Many ty URN103L . Esculinhamnosus , Leuconoteroides , and Laclantarum , among LL. buchneri group is located within the family Lactobacillaceae. This group contained only obligate heterofermentative Lactobacilli. The selected eight types of colonies with an esculin-positive reaction were analyzed by 16S rDNA sequencing to determine which species would be matched with strain URN103L, which was selected as the LAB strain among the 14 isolated strains with β-glucosidase activity. The DNA sequence of strain URN103L was 1474 bp, which was compared with the sequences in the NCBI database using BLAST. Phylogenetic trees of strain URN103L were downloaded from the NCBI database and modified in Adobe Illustrator , the supernatant of the crude extract containing β-glucosidase from s of TLC  and HPLCs of TLC . Clearlys of TLC  revealedLeuconostoc mensenteroides WiKim19 and Pediococcus pentosaceus WiKim20 with β-glucosidase activity isolated from kimchi had a greater ability to transform ginsenoside Rb1 into ginsenosides Rg3 and Rg5 than the other strains [Lactobacillus plantarum CRNB22 with β-glucosidase activity isolated from kimchi converted ginsenoside Rb1 into ginsenosides Rg3-s, Rg3-r, and Rg5 [Leuconostoc citreum can convert ginsenoside Rb1 in the following sequence: Rb1→Rd→F2→compound K [D. anomala YAE-1 fully converted ginsenoside Rb1 to ginsenoside Rd after 48 h of incubation [Although minor ginsenosides have various beneficial effects, only a small quantity is produced by enzymatic transformation in ginseng. Therefore, many studies have investigated enzymatic transformation methods using various beneficial microorganisms for conversion of major ginsenoside Rb1. It has been reported that various lactic acid bacteria isolated and identified from kimchi, a traditional Korean fermented food, can perform enzymatic biotransformation of ginsenoside.  strains . In addi and Rg5 . Dependi and Rg5 . Additiompound K . Consistcubation .L. buchneri URN103L was inoculated at 3%. The initial viable cell count just after inoculation was approximately 6.04 ± 0.077 log colony-forming unit (CFU) mL−1. After 7 days of fermentation, the viable cell count increased to 8.27 ± 0.044 log CFU mL−1 and decreased by 7.39 ± 0.083 log CFU mL−1 over 14 days. The six types of LAB in fermented ginseng grew by approximately 9~11 log CFU mL−1 on the first day, and after 5 days, they decreased to 6.0~7.5 log CFU mL−1 [L. buchneri URN103L converted major ginsenoside Rb1 to minor ginsenosides Rd and Rg3 (epimer R/S), as shown in L. buchneri URN103L in 20% ginseng root was incubated, ginsenosides Rb1 and Rd were metabolized into Rg3 after 3 days , has been shown to inhibit tumor growth. The most affected pathway of anti-human colorectal cancer activity is the Ephrin receptor pathway in HCT-116 human colorectal cancer cells [Lentilactobacillus buchneri URN103L with β-glucosidase activity, isolated from the fermentation of medicinal plants, has the potential to convert ginsenoside Rb1 into Rd and Rg3. In the future, we hope that various biological functions of ginsenosides, such as ginsenoside Rg3, will be revealed and play an important role in human health.Ginsenoside Rg3, one of the saponins in American ginseng  showed high hydrolytic activity on ginsenoside Rb1. The LAB strain was identified as


Results from immunofluorescence supported that oxyresveratrol effectively inhibited AKT and GSK3-β activation in individual cells upon TNF-α stimulation. Furthermore, functional assay confirmed that oxyresveratrol repressed the expansion of the HaCaT colony over 3 days, and this was caused by the ability of oxyresveratrol to induce cell cycle arrest at S and G2/M phases and the reduction in the expression of a proliferative marker (Ki-67) and a survival marker (MCL-1). Given the importance of TNF-α and the PI3K/AKT pathway in the psoriatic phenotype, we anticipate that oxyresveratrol, which targets the TNF-α-stimulated PI3K/AKT pathway, would represent a promising psoriasis therapy in the near future.Psoriasis is a complex inflammatory disease characterized by hyperproliferative keratinocyte caused by active PI3K/AKT signaling. TNF-α concentrated in the psoriatic lesions stimulates AKT activation. We previously discovered that oxyresveratrol inhibited inflammation via suppressing AKT phosphorylation, therefore oxyresveratrol may possess a conserved property to block AKT activation and proliferation in keratinocyte in response to TNF-α. Our current study proved that oxyresveratrol exhibited potent anti-proliferative effects against TNF-α. These effects are explained by the findings that oxyresveratrol could potentially inhibit TNF-α-stimulated AKT and GSK3- Psoriasis is well established to be an incurable chronic inflammatory dermatosis that impacts people around the globe ,2. The oAccumulated studies have revealed that proliferation of human keratinocytes is dependent on the PI3K/AKT signaling pathway . Many stOver the past decade, it has been reported that some small molecules specifically inhibiting the PI3K/AKT signaling provide great outcomes for psoriatic arthritis, and this may be an effective management for psoriasis . Studiestrans-2,3′,4,5′-tetrahydroxystilbene, OXY) is a natural stilbene that has lately gained prominence due to its simple chemical composition and varied pharmacological properties [Oxyresveratrol . Rabbit anti-phospho-AKT (Ser473) antibody, mouse anti-AKT antibody, rabbit anti-phospho-GSK3-β (Ser9), mouse anti-GSK3-β antibody, rabbit anti-Ki-67 antibody, rabbit anti-MCL-1 antibody, DAPI , and LY294002 (a specific inhibitor of the PI3K/AKT) were purchased from Cell Signaling Technology . Goat anti-mouse IgG-IRDye®800CW and goat anti-rabbit IgG-IRDye®680RT were purchased from Li-COR Biosciences . Goat anti-rabbit conjugated with Alexa488 was purchased from Thermo Fisher Scientific , MA, USA).Human immortalized keratinocytes (HaCaT) were purchased from Cell Lines Service GmbH . Recombinant human TNF-α was purchased from PeproTech . Oxyresveratrol (OXY) was purchased from Sigma-Aldrich . MTT reagent -2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich . Guava2 at 5%. The medium was changed every three days until the culture reached approximately 70% confluent. Cells were subcultured when they were 90% confluence. To determine the effect of TNF-α on cell number and cell cycle progress of HaCaT cells, different final concentrations (20–100 ng/mL) of TNF-α in DMEM, without fetal bovine serum, were added to HaCaT cells for 48 h. For other experiments, the highest concentration at 100 ng/mL of TNF-α was utilized.HaCaT cells were cultured with Dulbecco’s modified Eagle’s media (DMEM) , containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin , and incubated in an incubator set for proper humidity, temperature at 37 °C, and CO4 cells/well in DMEM were seeded in 96-well plates and incubated for 24 h. Then, cells were treated with varied concentrations of oxyresveratrol (OXY) with or without the existence of 100 ng/mL of TNF-α for 48 h. Cells were washed once with 1× PBS and incubated with 200 μL of the medium containing MTT reagent at the final concentration of 0.5 mg/mL for 1 h. Next, the MTT reagent was discarded, and each well was washed once with 1× PBS before adding 100 μL of 100% DMSO. Colorimetric measurement at 570 nm was performed using a microplate reader .HaCaT cells at 1 × 105 cells/well in DMEM were seeded in 24-well plates and cultured for 24 h. The medium was replaced with FBS-free medium, and the cells were treated with OXY at different concentrations  with or without the presence of 100 ng/mL of TNF-α. Incubation time was designed to cover 3 days; cells were collected and counted at 0, 24, 48, and 96 h. Cell collection was performed by washing cells with 1× PBS and treating cells with 1× trypsin solution (100 µL) for 10 min before adding 400 µL of complete medium (DMEM with FBS). Cell suspension in each well was counted for three times by using the CellDrop™ Automated Cell Counter .HaCaT cells at a density of 4 × 106 cells/well and cultured for 24 h. The medium was replaced by adding 2 mL of FBS-free medium, and cells were cultured for 24 h. Then, cells were treated with OXY at 40 µM for 3 h, before stimulating with 100 ng/mL of TNF-α. Cell lysates were collected at different time points  by adding 300 µL of 1× reducing Laemmli buffer. Next, the cell lysates were heated at 95 °C for 5 min, separated by SDS-PAGE using 10% gel, and transferred onto polyvinylidene difluoride (PVDF) membranes . After blocking with blocking solution  in TBST) for 1 h at room temperature (RT), membranes were incubated overnight with 1:1000 (diluted in blocking solution) of antibodies against phosphorylated Ser473 of AKT, total AKT, phosphorylated Ser9 of GSK3-β, and total GSK3-β. Membranes were washed trice with TBST (5 min each time), then membranes were incubated with proper secondary antibodies for 1 h. After washing membranes for 5 times with TBST, immunoreactive bands were visualized and recorded with the Odyssey® CLx Imaging System . The band intensity was analyzed and quantified using the ImageJ software. For additional experiments to assay a dose-dependent effect of OXY, we performed similar procedures, but the difference was that we varied concentrations of OXY; 10, 20, and 40 µM were examined. Moreover, an inhibitor of PI3K, LY294002, at 10 µM was included. Based on the results from Western blot where it showed that phosphorylation of AKT (pAKT) and GSK3-β (p-GSK3-β) was peaked at 40, and 20 min after TNF-α stimulation, respectively, the harvesting time point for detecting pAKT was performed at 40 min after TNF-α stimulation, whereas the time for detecting GSK3-β was at selected at 20 min post- TNF-α stimulation.HaCaT cells in DMEM were seeded in 3-cm dishes a density of 0.5 × 106 cells/well in DMEM were seeded onto glass coverslips placed in 3-cm dishes and cultured for 24 h. The medium was then replaced by FBS-free DMEM, and cells were cultured for 24 h. Cells were pre-treated with OXY at 40 µM for 3 h before stimulating with TNF-α for 40 min (for pAKT staining) and 20 min (for p-GSK3-β staining). For Ki-67 and MCL-1, the OXY-treated cells were stimulated with TNF-α for 24 h. Cells were washed once with 1× PBS and fixed with 4% paraformaldehyde for 15 min. Sample coverslips were washed 3 times with PBS (5 min each time) and permeabilized with 0.3% TritonX-100 in PBS for 5 min. After washing trice, sample coverslips were blocked with 1% BSA in PBS for 1 h at RT, and incubated with primary antibodies in a moist chamber at 4 °C overnight. Sample coverslips were washed 3 times with 1× PBS and incubated with a secondary antibody conjugated with Alexa488 for 2 h in a moist chamber at RT. Sample coverslips were washed 3 times with 1× PBS and 1 time with deionized water (5 min) before mounting with Fluoromount-G . Signals of the target proteins were visualized at 100× magnification by a fluorescent microscope, Axio Vert.A1 . The Zen 2.6 (blue edition) software for the Zeiss Axiocam 506 color microscope camera was used to capture and analyze the images.HaCaT cells at a density of 0.5 × 104 cells/well. Cell suspension was gently added into each well to create single cells with homogeneous cell distribution and cultured for 24 h. The medium was changed to an FBS-free medium, and cells were pre-treated with various concentrations of OXY  for 3 h. Cells were then stimulated with 100 ng/mL of TNF-α. The size of the HaCaT colony at 0, 24, 48, and 96 h post-treatment was monitored and captured by an Axio Vert.A1 microscope  equipped with the Zen 2.6 (blue edition) Software for the Zeiss Axiocam 506 color microscope camera. In addition, cell counting was performed after 96 h to verify the association of the colony size and the cell number.HaCaT cells were seeded in 3-cm dishes in DMEM at a low density of 5 × 105 cells/well in DMEM for 24 h. The medium was replaced by a serum-free medium, and cells were cultured for 24 h. Next, cells were pre-treated with OXY at different concentrations  for 3 h and stimulated with 100 ng/mL of TNF-α for 48 h. Culture medium was discarded and cells were rinsed with 1× PBS for 3 times. Cells were collected by trypsinization for 10 min and washed once with 1× PBS by centrifugation. Cells were then fixed and permeabilized by 70% ice-cold ethanol in a dropwise fashion to prevent cell aggregation. Cells were fixed for 24 h at −20 °C overnight. Cells were washed with 1× PBS for 1 time by centrifugation, and 150 µL of Guava® Cell Cycle Reagent was added to each group of cells and incubated in the dark for 15 min at RT. Stained cells were analyzed immediately by flow cytometry using a flow cytometer DxFLEX from Beckman Coulter . Data analysis was performed using the CytExpert for DxFLEX software, and results were presented as the ratio of cell population in G1, S, and G2/M phases.Cells were seeded in 24-well plates at a density of 4 × 10p < 0.05. All experiments were repeated at least three times independently.Data are presented as mean ± standard error of mean (SEM). Differences between groups were analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple comparisons on RAW data reads. Significant differences compared with appropriate controls are denoted with asterisks, * 5 cells/mL, TNF-α at 20 ng/mL did not create any significant change (2.95 ± 0.01 × 105 cells/mL)  for 48 h were directly counted. Results showed that in comparison to the untreated HaCaT cells where the cell count was 3.14 ± 0.46 × 10ells/mL) A. Howeveectively A. This dectively B. Since We further evaluated whether OXY has inhibitory effects against the proliferative influence of TNF-α. We first performed an MTT assay to examine the viability (which indirectly reflects proliferation) of HaCaT cells treated with various concentrations of OXY, ranging from 0 to 160 µM, in combination with 100 ng/mL TNF-α. We found that HaCaT cells treated with TNF-α alone exhibited a significant increase in cell viability to 160% compared to the untreated group A. Intereβ. When the cells were stimulated with TNF-α, phosphorylation of AKT was rapidly started at 10 min and gradually increased over the course of 210 min  throughout the course of 210 min  and an anti-apoptotic protein (MCL-1), which is one of the cell survival markers. Data from immunofluorescence study obviously presented high expression of Ki-67 in the nuclei of TNF-α-stimulated cells , verifying that OXY authentically blocks the PI3K/AKT signaling pathway in response to TNF-α induction. We further investigated, by performing functional tests, to understand how OXY interferes the proliferative effect of TNF-α. A colony expansion assay clearly showed that OXY blocked the colony forming of HaCaT cells, and this was related to the reduced number of cells over the period of 96 h. Moreover, we finally discovered that OXY significantly induced cell cycle arrest at S and G2/M phases in a pattern similar to that of the PI3K inhibitor. It is reasonable to obtain this phenomena as a result of suppressing the TNF-α-induced PI3K/AKT signaling pathway by OXY since it is well defined that phosphorylated AKT controls the intracellular functions of its substrates related to cell cycle progression at the G1/S and G2/M transitions [The observation that TNF-α stimulated HaCaT cell proliferation directs us to realize the role of the PI3K/AKT signaling pathway since this particular signaling pathway creates an intracellular network for the regulation of cell proliferation . Specifinsitions . To be mnsitions . As antinsitions  or rationsitions  should bOn the basis that during the development of psoriasis, responsible immune cells (T cells) extensively secrete TNF-α, which then play a significant role in the pathogenic process , there h


Phase investigation of Li3N, FeN2, and Li3FeN2 was carried out. The discharge capacity of Li3FeN2 is 343 mAh g−1, which is about 44.7% of the theoretic capacity. The ternary nitride Li3FeN2 molar heat capacity is calculated using the formula p,mC = 77.831 + 0.130 × T − 6289 × T−2, . The thermodynamic characteristics of Li3FeN2 have the following values: entropy S0298 = 116.2 J mol−1 K−1, molar enthalpy of dissolution ΔdHLFN = −206.537 ± 2.8 kJ mol−1, the standard enthalpy of formation ΔfH0 = −291.331 ± 5.7 kJ mol−1, entropy S0298 = 113.2 J mol−1 K−1 (Neumann–Kopp rule) and 116.2 J mol−1 K−1 (W. Herz rule), the standard Gibbs free energy of formation ΔfG0298 = −276.7 kJ mol−1.Li In the world of technological development, energy sources are being severely depleted. In this regard, the issues related to creating new energy sources, in particular renewable energy sources, are being considered.Secondary batteries, such as lithium-ion, lithium sulfur, and hydrogen batteries, are attracting increased attention for their development and production. Probably, one of the prospective renewable sources of energy is the lithium-ion battery (LIB) as an energy source for many applications, such as electric cars and buses, laptops, mobile phones, etc. LIBs solve the problems of high energy requirements , environmental efforts, and relatively low cost.A lot of efforts were directed to the development of more advanced batteries. For example, different approaches for LIB’s development were used, such as nanostructured materials ,11,12,13−1), although the large volume expansion of silicon during the charge/discharge process (300%) leads to a capacity decrease and reduced cycle life [Thus, a lot of efforts were focused on the fabrication of anode materials with high theoretical specific capacity. For example, silicon has attracted the attention of the LIBs industry as an anode material with ultrahigh specific capacity , and a low theoretical capacity of 175 mAh g−1 [Another popular anode material with high performance is Lindliness ,59. Howe mAh g−1 ,61,62,633N-type anodes, e.g., Li2Na4N2 and Li4Na2N2 phases [3N-Mg3N2 [2n-1MN [3FeN2 [3FeN2 materials have transition metal, it could not be used as solid electrolyte because transition metals might produce conduction electrons, which is unacceptable for a solid electrolyte of lithium battery. However, this quality is advantageous for using this material as an electrode. Li3-xFeN2 (0 < x < 1) has a high capacity of 260 mAh g−1 [Previous works shows good electrochemical properties of Li2 phases , LiBeN [2 phases , Li3N-Mg3N-Mg3N2 , Li2n-1M [2n-1MN , and Li3N [3FeN2 ,69. Thus mAh g−1 . In addi3FeN2 was first synthesized by Frankenburger et al. by the reaction of lithium nitride (Li3N) with elemental Fe in N2 atmosphere [3N with iron using thermogravimetry [3FeN2 was indexed by an orthorhombic cell with lattice parameters a = 9.65 Å, b = 8.66 Å, and c = 8.38 Å. Emery et al. [3N with Fe powder in atmosphere, which shows a cationic mixing in Li3FeN2 compound. Limosphere . After davimetry . These sy et al.  show the3FeN2 is a prospective material for hydrogen storage because of its hydrogen uptake capacity of 2.7 wt %, of which about 1.5 wt % was reversible [Liversible ,72,73.3FeN2 are shown. Firstly, Li3N synthesis was obtained in an adiabatic chamber. Then, mixed with iron nanopowder, Li3FeN2 was obtained at a tube furnace. Two-step synthesis was chosen for the synthesis of high-purity complex nitride Li3FeN2.In this article, the two-step synthesis and properties of promising anode material Li3) were used as starting components for Li3N, Fe2N, and Li3FeN2 synthesis . A 16 mm diameter and 0.6 mm lithium plate sliced and polished in an argon glovebox, iron nanopowder, nitrogen, and ammonia . The tube has been purged by ammonia (NH3) for 30 min before synthesis. Synthesis was carried out in NH3 atmosphere at 530 °C for 6 h with a heat rate of 8 °C/min. Mechanically mixed and powder was hot pressed for 2 h at 1100 °C. The received hot-pressed sample was heated in N2 atmosphere for 10 h at 700 °C (heat rate was 5 °C/min). After heat treatment, the sample was mechanically ground into ivory-colored powder.Iron nanopowder and nitrogen were used as a source for FeXRD analysis was held with a Bruker D8 Advance  with a step of 0.02°. Structural parameters were refined by the Rietveld method using TOPAS5 software.α-radiation. The diffraction angles (2θ) are 5–100°, 10–80°, and 5–120° for Li3N, Fe2N, and Li3FeN2, respectively.X-ray diffraction analysis (XRD) was used as the structure analysis method for the synthesized nitrides powders investigated. XRD analysis was performed with a Bruker D8 ADVANCE diffractometer with a vertical goniometer and Cu K−3 HCl was used for the calorimetric cell ampoule. The ampoule was broken when thermal equilibrium was established, and nitride powder began to dissolve in HCl solution. Thermo-EMF vs. time was registered during the dissolution process providing the heat dissolution curve. Integration of this curve gave the value of dissolution enthalpy.Calorimetric measurements were performed using a TAM IV Microcalorimeter  at 298 K with the cell volume of 20 mL. Aqueous solution of 1 mol dm3N (a) and Fe2N (b) powders. All peaks are in good correlation with database one. Li3N has a P6/mmm space group with lattice parameters a = 3.6711 Å, b = 3.6711 Å, and c = 3.8770 Å, which are in good correlation with [2N reflection peaks also are in good correlation with [2N is P312 with lattice parameters a = 4.7912 Å, b = 4.7912 Å, and c = 4.416 Å.ion with  and PDF ion with  and PDF 3N, Fe2N, and Li3FeN2 after heat treatment in an Netzsch APTAK chamber, tube furnace with ammonia atmosphere, and tube furnace with nitrogen atmosphere, respectively. Lattice parameters, a and c, calculated by the Rietveld method for Li3FeN2 are a = 4.872 Å, b = 9.677 Å, and c = 4.792 Å, respectively, in the Ibam space group. XRD patterns of Li3FeN2 synthesized at different temperatures are shown in 2O impurity. Other samples include such impurities as Li2O (PDF #01-076-9237), Li5FeO4 (PDF #01-075-1253), and LiFeO2 (PDF #74-2284). Samples synthesized at 850 °C have only Li2O impurity; thus, further investigation of the compounds were conducted with materials synthesized at 850 °C.+ is in 4b and 8g, Fe+3 is in 4a, and N−3 is in 8j sites. All calculations were carried out with using TOPAS 4 software by Bruker. The final structure parameters (including site occupancy) are listed in The structure refinement defined that Li3FeN2 (LFN) compound from single nitrides Li3N and Fe2N is calculated using the following equation (ΔoxHLFN):The formation enthalpy of LidHLFN) has been determined in the calorimeter. The received value was equal to −1972.96 ± 25 J g−1, as shown in For enthalpy calculation, we used thermodynamic cycle with the following reactions, as shown in oxHLFN is obtained by the next equation:The resulting value of ΔdHLi3N, ΔdHFe2N, and ΔdHN2 were also measured by the calorimetry method. Measurement results are shown in oxHLFN by Equation (8) is equal to −94.833 kJ mol−1. The negative value of ΔoxHLFN defines Li3FeN2 as a stable phase. In addition, it is it is energetically favorable to synthesize LFN from single nitrides.The values of Δ3FeN2 from elements can now be calculated using the following equation:At last, the enthalpy of formation of LiStandard enthalpies for the calculation were taken from the handbooks ,77, as s3FeN2 formation by Equation (9) is −291.331 ± 5.7 kJ mol−1, fH0 for Li3FeN2 has the same order as for similar compounds, namely lithium metal nitrides , and m—a mass. The sample weight consists of synthesized compound (Li3FeN2) and impurity (Li2O). So, the heat capacity of Li3FeN2) is expressed from Equation (10) as:The temperature dependence of the isobaric heat capacity of the Li2O):The weight of the included compounds can be found from the total mass of the sample, which are calculated through the weight fraction of lithium oxide, ω(LiAccording to Equations (12) and (13), Equation (11) can be written as follows:−1 K−1, b = –13.63·10−3 J mol−1 K−2, and c = –18.624·105 J mol−1 K. The heat capacity of Li3FeN2 for the 300–900 K temperature range was recalculated using Equations (15) and (16) considering Li2O’s impurity presence. According to XRD data  can be written as (according to Equation (1)):Thereby, the heat capacity of LFN can be calculated from the experimental data and heat capacity of lithium oxide impurity. For Equation (14), it is required to know the dependence of the specific heat capacity of the lithium oxide from temperature. For this, tabulated data for the lithium oxide heat capacity  were useXRD data , Li3FeN2The dependence of the heat capacity by temperature calculated from Equation (17) using tabular data  is shown2O impurity amount). However, XRD quantitative analysis gives rough results for the small presence of compounds in the material. For other quantitative methods, the amount of impurities can be measured more accurately: for example, thermogravimetry or volumetric methods.The temperature dependence of the heat capacity calculated by the Neumann–Kopp rule is in good correlation with the recalculated heat capacity . Since the entropy can be calculated by the Neumann–Kopp rule, if there is no phase transition until the calculation temperature, entropy can be also calculated by the Neumann–Kopp rule:Entropy is another thermodynamic function that should be calculated. The Third Law of thermodynamics states, “The entropy of a perfect crystal is zero when the temperature of the crystal is equal to absolute zero (0 K).”. Thus, the entropy absolute value can be valued by the equation:3FeN2 at room temperature is 113.2 J mol−1 K−1 according to Equation (20) and tabular data [3N is 27.2 cm3 mol−1 , for Fe5 g cm−3 ), and fo9 g cm−3 ). The suH is Herz constant (KH = 20.5), M is molar mass, Cp,298 is isobaric heat capacity, and m is atoms per formula. According to Equation (21) and considering Cp,298 from −1 K−1. Thus, the LFN entropy calculated by the Herz rule is in good correlation with the Neumann–Kopp rule result.In addition, the LFN entropy can be calculated by the W. Herz rule :(21)S2983FeN2 formation (at T = 298 K):The enthalpy of formation and entropy calculated above allows evaluating the standard Gibbs free energy of Li3FeN2 at room temperature is −276.7 kJ mol−1.The resulting value of the Gibbs free energy for LiThe next reaction is suggested for the determination of stability against metallic lithium with subsequent calculation of the Gibbs free energy at room temperature:3N, it is −128.6 kJ mol−1 [3FeN2 Gibbs free energy has been calculated above. Thus, the Gibbs free energy for reaction (23) is 19.5 kJ mol−1, and this reaction is thermodynamically impossible. Finally, Li3FeN2 is stable against metallic lithium at room temperature.To determine the Gibbs free energy of the reaction, it is required to subtract from kJ mol−1 . The Li33FeN2 anode material for a lithium-ion battery. The two-step synthesis method allowed producing a highly pure compound with less than 3 wt % of Li2O impurity according to XRD data. The enthalpy of Li3FeN2 formation from binary nitrides was determined according to the measured enthalpy of dissolution of reagents and product of Li3FeN2 formation reaction. The obtained value is equal to −206.5 ± 2.8 kJ mol−1. The Li3FeN2 standard enthalpy of formation from single elements is equal to −291.3 ± 5.7 kJ mol−1. This value can be used in further thermodynamic modeling and determinations.The thermodynamic characteristics were determined for Li2O impurity. The temperature dependence of the heat capacity is in good correlation with calculation by the Neumann–Kopp rule. Finally, the heat capacity can be described by formula Cp(T) = 78.997 + 0.132 × T + 4.654·105 × T−2, where T is absolute temperature. The LFN entropy is equal to 113.2 J mol−1 K−1, and the Gibbs free energy of Li3FeN2 formation is −276.7 kJ mol−1. The calculations confirm that the Li3FeN2 material is stable against metallic lithium. All thermodynamic values and functions can be used for modeling and further calculations.The heat capacity value was recalculated considering the presence of Li


In this work, the effects of rising atmospheric CO2 levels on the metabolic active soil microbiome have been investigated at the Giessen free-air CO2 enrichment (Gi-FACE) experiment on a permanent grassland site near Giessen, Germany. The aim was to assess the effects of increased C supply into the soil, due to elevated CO2, on the active soil microbiome composition. RNA extraction and 16S rRNA (cDNA) metabarcoding sequencing were performed from bulk and rhizosphere soils, and the obtained data were processed for a compositional data analysis calculating diversity indices and differential abundance analyses. The structure of the metabolic active microbiome in the rhizospheric soil showed a clear separation between elevated and ambient CO2 (p = 0.002); increased atmospheric CO2 concentration exerted a significant influence on the microbiomes differentiation (p = 0.01). In contrast, elevated CO2 had no major influence on the structure of the bulk soil microbiome (p = 0.097). Differential abundance results demonstrated that 42 bacterial genera were stimulated under elevated CO2. The RNA-based metabarcoding approach used in this research showed that the ongoing atmospheric CO2 increase of climate change will significantly shift the microbiome structure in the rhizosphere.Elevated levels of atmospheric COThe online version contains supplementary material available at 10.1007/s00248-021-01791-y. Total annual anthropogenic greenhouse gas emissions have continued to increase, comprising CO2, which represents around 75% of these emissions  sodium dodecyl sulfate) were added, and the disruption process was performed again as described above. Both supernatants from the lysis processes were pooled in one reaction tube.RNA extraction was performed following a modified protocol of Mettel et al. . For theThe pooled supernatant was immediately extracted, initially with the addition of 500 µl of phenol/chloroform/isoamyl alcohol (25:24:1) and subsequent with chloroform/isoamyl alcohol (24:1). Afterwards, each time the sample was centrifuged for 5 min at 17,000 g and 4 °C. The resulting upper aqueous phase was transferred to a new reaction tube, 800 µl of PEG solution was added (30% [wt/vol] polyethylene glycol 6000 and 1.6 M NaCl), incubated in ice for 30 min and centrifuged for 30 min at 17,000 g and 4 °C. Subsequently, the DNA/RNA pellet was washed with 800 µl of ice-cold 75% ethanol, dried out and dissolved in 50 µl of nuclease free water.After extraction, samples were treated for DNA digestion with RNase-Free DNase Set (QIAGEN GmbH — Germany) according to manufacturer instructions; DNase reaction was stopped with 10 µl of 50 mM EDTA. With the DNA-free RNA, a PCR was carried out, using the universal 16S rRNA gene primers 27F (5’-AGAGTTTGATCMTGGATCMTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’) , 49 and The 16S rRNA gene hypervariable regions (V4&V5) were PCR amplified using the set of primers 520F (5’-AYTGGGYDTAAAGNG-3’)  and 907R2 conditions and soil habitats using the Wilcoxon test , eCO2 decreased 16S rRNA gene abundance in rhizosphere soil by 31%. Also, Marhan et al. [2 but by sampling date and depth. Likewise, Brenzinger et al. [2 and eCO2 rings were found regarding the 16S rRNA gene. However, Bei et al. [2 conditions the functional metagenome of rhizosphere soil presented an increase on amino acids and carbohydrates metabolisms, membrane transporters, and quorum sensing proteins: similar to our study’s PICRUSt2 results (S5).Greater carbon input due to eCOg et al.  describeg et al.  showed ta et al.  demonstra et al. , who repn et al.  describer et al.  and Bei r et al.  reportedi et al.  describe2 conditions; among these are Pseudomonas and Bacillus  and a Pseudomonas strain (AS1) displayed high potential for lignocellulose decomposition due to a variety of cellulase and xylanase activities. Trujillo-Cabrera et al. [Bacillus thuringiensis and Pseudomonas gessardii. The augmentation of these taxa would agree with the predicted functional metagenome, which indicated an increment of several enzymes involved in lignocellulose materials degradation, as Chitinase (EC:3.2.1.14), Endo-1,3(4)-beta-glucanase (EC:3.2.1.6), Endo-1,4-beta-xylanase (EC:3.2.1.8), and Cellulase (EC:3.2.1.4) (S5). Similar results have been described by He et al. [2 in soybean and maize fields. The above-mentioned increase of C compounds degradation could occur as a response of greater C availability due to an increase of root exudates under eCO2 conditions.In addition, several genera involved in the degradation of carbon (C) compounds were stimulated under eCOlus Fig. , S2 thatradation . Maki etradation  reporteda et al.  describee et al. , 19, whoe et al.  describe3−, available K+, soil microbial biomass carbon (SMBC), and available PO42− [2 rings in comparison with ambient ones and might also explain why some genera involved in different processes of the nitrogen cycle were stimulated under eCO2 conditions. Genera belonging to families Rhizobiaceae and Xanthobacteraceae as Rhizobium, Mesorhizobium, and Phyllobacterium have been extensively reported as nitrogen-fixing bacteria [2 fold increases ranging from 6.78 to 8.04. Also, PICRUSt2 results indicate that functional orthologs of the enzyme nitrogenase (EC:1.18.6.1) were significantly augmented in eCO2 rhizosphere soil (S5). This increase in the abundance of nitrogen-fixing bacteria could have occurred as response to N deficiency, which eventually became a limiting factor for biomass production under eCO2. Similar results were reported by Li et al. [15 N in mine tailing soils under eCO2 and a dominance of uncultured nitrogen-fixing bacteria.Moreover, these changes in the C influx could induce a reduction of available N in the soil ecosystem , which ale PO42− . The afobacteria –90, and i et al. , who des4+ content and Nitrospira genus under eCO2 conditions. Although NH4+ values were not significantly different between aCO2 and eCO2, NH4+ content was on average 10% higher in aCO2 soils, which suggest that nitrification processes could have been affected due to elevated environmental CO2. Alterations in nitrification process in the Gi-FACE have been already described by Müller et al. [2 reduced NH4+ oxidation to NO3− by 25%.Aldex2 correlation results demonstrated a significant negative correlation between NHr et al. , who repStreptomyces, Rhodanobacter, Pseudomonas, Burkholderia, and Bacillus were significantly stimulated in eCO2 rhizospheric soil with Log2 fold changes between 5.37 and 8.065 [2 conditions (S5).Furthermore, several denitrifying genera as nd 8.065 –96. Addi2 concentrations alongside other environmental factors. The increment of carbon input due to eCO2 possibly augmented labile carbon degradation in rhizosphere soil reflected by the increment of bacteria biomass and CO2 soil emissions. The aforementioned processes could cause a nitrogen constraint, observed in the increment of the C:N ratio, and decreased of NH4+, which likely triggered a shift in the rhizosphere soil microbiome with an increment of nitrogen fixing and denitrifying taxa. The observed increase of denitrifier genera might explain the increased N2O fluxes under eCO2 conditions, previously described in the Giessen FACE [2 conditions, N2O emissions were 1.79-fold higher and that the linear interpolations showed a 2.09-fold increase in N2O emissions mostly because of the oxidation of organic N and incomplete reduction of NO2−, emitting N2O instead of N2 Supplementary file2 (XLSX 101 KB)Supplementary file3 (XLSX 73 KB)Supplementary file4 (DOCX 891 KB)Supplementary file5 (XLSX 649 KB)Below is the link to the electronic supplementary material.


Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos , thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts.Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-TyrThe online version contains supplementary material available at 10.1007/s11248-021-00248-9. The knockout (KO) mouse model plays a critical role in the new era of functional genomics and is widely used to study the role of specific genes. A global scientific collaborative initiative, the International Knockout Mouse Consortium (IKMC), planned to use C57BL/6N-derived ES cells to generate KO models for every mouse gene. As a result, a repository of mutant mES cells has been established to serve the global research community. Based on a collaboration, our center obtained this repository and is now acting to serve the research community in the Asia-Pacifc region. We therefore hold the responsibility to distribute the resource and also to generate as many more mouse models as possible using the mES cells.There have been several developments that aim to improve the efficiency of generating a genetically modified model using mES cells. Morula aggregation and 8-cell coculture are both accessible techniques that can be used to produce germline transmittable chimeras from C57BL/6N (B6N) ES cells; these techniques are cost effective and do not require extensive training  in the specific-pathogen free (SPF) facility at Soochow University. All experiments were carried out according to regulatory guidelines for experimental animals approved by the Institutional Animal Care and Use Committee (IACUC) of CAM-SU Genomic Resource Center.B6NTac and B6 albino females (4–5 weeks-of-age) were injected intraperitoneally with 5 IU/0.1 ml pregnant mare serum gonadotropin . Then 46–48 h later, these mice were injected with 5 IU/0.1 ml human chorionic gonadotropin . Females were subsequently mated with the same fertile male strains. Embryos were collected at E2.5 by flushing oviduct with M2 medium  and cultured in M16 medium  under mineral oil  at 37 °C under 5% CO2.Genetically modified B6N ES cell lines were generated by IKMC and available at the CAM-SU Genomic Resource Center, Soochow University. ES cell clones from the primary frozen archive were thawed and seeded onto feeders in M10 medium  supplemented with 10% fetal bovine serum, 2 mM L-glutamine , 1 × penicillin/streptomycin, 0.1 mM ß-mercaptoethanol and 1000 U/ml leukemia inhibitory factor ) for 2–3 passages in the presence of G418. All cell clones used for microinjection were karyotyped by qPCR using Taqman probes for chromosomes 1, 8, 11 and Y.B6N-derived ES cells were thawed 3 days before injection and cultured in M10 medium in the presence of 2i inhibitors (1 μM PD0325901 and 3 μM CT99021), passaged once when they reached 70–80% confluency and switched back to M15 medium  supplemented with 18% fetal bovine serum, 2 mM L-glutamine , 1 × penicillin/streptomycin, 0.1 mM ß-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor  on the day prior to microinjection. On the day of injection, ES cells were trypsinized and switched back to microinjection media (M15 + HEPES). These cells were kept on ice whilst transferring for microinjection.ES cells were injected into blastocysts using an inverted microscope .Approximately 10–15 ES cells with small, round and smooth morphology were injected into each blastocyst. After 2–3 h recovery, 7–8 injected blastocysts were transferred into each uterine horn of a 2.5 dpc 8- to 12-week-old psedopregnant ICR female that had been anesthetized by the intraperitoneal injection of Avertin (0.4 mg/g body weight).Chimeric mice were identified by coat color at weaning. The combination of JM8A3.N1 ES cells with B6NTac host embryos resulted in agouti/black chimeric mice while the B6 albino host embryos resulted in agouti/white chimeras. At 21 days after birth, the number, sex, and extent of coat color chimerism, was recorded in the offspring. Three male chimeras (6–8 weeks-of-age) with the highest contribution of coat color were chosen as breeders. One male chimera was mated with two B6NTac females. Germ-line transmission in the G1 offspring was confirmed by PCR and subsequent sequencing of the PCR fragment.p < 0.05 was considered to be statistically significant.Statistical analyses were carried out by using GraphPad Prism version 8.0.2 . Data are presented as mean ± SD. Differences were compared by the t-test and P < 0.0001) . However, the live birth rate was significantly higher for B6NTac host embryos than for the B6 albino embryos   and a significantly higher percentage of male chimeras with more than 40% agouti coat color (12.4% vs. 9.4%). In addition, there was a significantly higher percentage of full colored mice (100% agouti mice) in the B6NTac blastocysts than B6 albino blastocysts . Further study the percentage of chimera formation, when analyzed as a percentage of the number of live male chimeras, the distribution of coat color contribution for the B6 albino embryos ranged from 40 to 99%; this compared to 80–100% in B6NTac embryos  derived from 118 genes  were tested for GLT Table . Both hoKO mice are usually generated by the injection of genetically manipulated ES cells into blastocysts. Over recent years, there has been a significant increase in the use of B6N-derived ES cells to produce KO mice. Differing combinations of host embryos and ES cells leads to variation in the ability to produce KO mice Below is the link to the electronic supplementary material.


During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown.in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae.Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 circa 60% within 60 min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells.CCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis. •in vitro model of the blood-brain barrier (BBB).The chemokine CCL4 induced phosphorylation of P38 in an •CCL4 treatment resulted in reduction of plasma membrane VE-cadherin and junctional ZO-1.•in vitro and in vivo.CCL4 induced neurovascular barrier breakdown  EngagemChemokines regulate the recruitment and activation of circulating and resident immune cells in all tissues, including the brain, and orchestrate immune activity during acute and chronic neuroinflammation. The effect of some CCL chemokines on ECs has been explored, but the contributions of the CC chemokine CCL4 (previously known as MIP-1β) in particular, on BBB function, remains poorly understood. CCL4 overexpression in the brain has been reported in a number of diseases including MS , AD Zhu and PD , and as + T cells to activated brain ECs  coated cell culture plates/flasks and media was changed every other day following a DPBS wash. Cultures were grown to 75–90% confluence before passaging and were used between passage 26 and 35. Culture medium was changed every other day until cells reached 75–90% confluence for renewed passaging. This cell line has been extensively used as a model of the BBB in the context of neuroinflammation (HCMEC/D3 (passage 26 and 35)  were culammation .2.24 - 1x105 cells/mL in gelatine-coated cell culture plates or flasks and media was changed every other day. Cells were grown in a humidified atmosphere at 37 °C in 5% carbon dioxide (CO2) and up to 75–90% confluence and used between passage 2 and 5.HUVEC were cultured in EGM-2 medium supplemented with EGM-2 B BulletKit™ ) as indicated by the manufacturer. cells were seeded at 8x102.3Cells were lysed in lysis buffer . Samples were collected and denatured at 100 °C for 5 min and centrifuged and frozen at −20 °C until further use. Alternatively, cells were lysed in RIPA buffer with protease and phosphatase inhibitors (to avoid proteolysis and preserve phosphorylation of proteins), and centrifuged. Supernatants were further denatured using lysis buffer. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously described  using an2.42 Petri dishes and stimulated with CCL4 for 5, 10, or 60 min, fixed in 4% paraformaldehyde and blocked in PBSA (0.5% with sodium azide). Nuclei of cells were visualised by the addition of 1 μg/mL Hoechst 33258 (bis-benzminide) or DAPI in the secondary antibody staining solution. The dishes were stored at 4 °C, mounted, and imaged using the Zeiss LSM 710 confocal microscope. Optionally, cells were permeabilised using ice cold acetone at −20 °C. Cells were then incubated with primary and secondary antibody at 37 °C in a humidified chamber. To reveal surface VE-cadherin, permeabilisation was performed after the first primary antibody incubation. Anti-Human VE-Cadherin Monoclonal Antibody (R&D Systems) and VE-cadherin Antibody (C-19) (Santa Cruz Biotechnology) were used at 1:200, P–P38 MAPK (T180/Y182)  was used at 1:3000 and ZO-1 (Thermofisher) was used at 1:50, in 0.1% PBSA. Secondary antibodies were used at 1:300 in 0.1% PBSA.HCMEC/D3 were grown on collagen I coated 35 mm2.5hCMEC/D3 cells were grown on 12-mm transwell filters with 0.4 μm pore size. The technique was performed as described previously . 70 kDaRecombinant Human MIP-1β (CCL4)  was purchased from Peprotech (cat. Number 300-09). As per manufacturer's indication, purity is ≥ 98% by SDS-PAGE gel and HPLC analyses .2.6+ cells were isolated from peripheral blood from healthy volunteers using Ficoll® Plaque Premium and positive selection on CD4+ MACS beads (Miltenyi) as previously described . Video microscopy (1 frame per 2s) recorded the time-dependent loss of dye in a single vessel, occluded by a glass probe. A baseline recording was established for 20–60 s. To obtain measurements, the flow of superfusing solution was stopped so that a pool was formed between the brain and the microscope's water immersion lens. Dye was injected into the circulation and the probe was placed over the vessel to be analysed. Remaining dye in the circulation was removed by the blood flow. For the abluminal CCL4 additions, the compounds were applied to the pool on the brain surface. Image analysis and densitometry was performed using ImageJ 1.45s (NIH). At the end of the experiment animals were culled by an overdose of the anaesthetic.t-test was used to compare differences between the two time sets (before and after additions).The mean pixel intensity was measured for each vessel tested for each frame/2 s and plotted against time. Data was normalized to diameter of the vessel according to pre-set graticule that correlates vessel diameter in a 20x Olympus field (used for the experiment) with 10 μm grid. Mean pixel intensity before and after addition and corresponding SEM were calculated and a paired 2.9Experiments performed on adult C57BL/6J mice within guidelines set by The Animals (Scientific Procedures) Act 1986 C57BL/6J and the EU Directive 2010/63/EU guidelines for animal experiments.t-test for variance of mean values.Adult C57BL/6J animals were anaesthetised using an intra-peritoneal injection of Ketamine (60 mg/kg) and Domitor® (10 mg/kg). For intravitreal injections, 1 μL of CCL4 (at 1 mg/mL diluted in 0.1% PBSA) to give a final dose of 1 μg per eye. Control animals received 1 μL of vehicle. FFA was carried out 10 min following CCL4 injections using a Micron III: pupils were dilated with 1% tropicamide, eyes kept moist with Viscotears®, and light fundus images acquired followed by green fluorescence images at 1.5 and 7 min following subcutaneous injection of 2% fluorescein. Statistical analysis carried out using Student's 33.1The expression of CCR5, the main receptor for CCL4 , was exa3.2P38 mitogen-activated kinase is a key regulator of neurovascular barrier function . Stimula3.3In most cells, including ECs, p38 activation results in the induction of stress fibre formation . Stress Therefore, we next assessed whether CCL4 induced stress fibre formation. Strong stress fibres were observed in hCMEC/D3 following treatment with CCL4 A. The in3.4circa 30% and 60% at 10 and 60 min respectively, demonstrating that CCL4 has a profound effect on a key junctional protein  and total VE-cadherin see D, indica3.6in vivo were investigated. A similar, significant circa 1.5-fold increase in sulforhodamine permeability was also observed in Wistar rat pial vessels in response to CCL4 in vivo  and posttraumatic stress disorder (PTSD) are characterized by raised levels of inflammation, hypothalamic-pituitary-adrenal axis disfunction and reduced neurogenesis. Depressed individuals have shown to have lower levels of CCL4 than healthy individuals . In factAbnormal levels of chemokines are also found in MS patients. CCL4 has been established as a marker for MS and correlates well with disease duration . PertineCCL4 may also influence neurogenesis; an agonist against CCL4's receptor, CCR5, facilitated neurogenesis in seizure-induced injury in the CNS . These eAltogether, our results suggest a potential for CCL4 as a novel modulating factor in BBB endothelial cells.10.13039/100010269Wellcome Trust (090326) and the 10.13039/501100000833Rosetrees Trust/Stoneygate Trust (CM 3001).This work was funded by grants awarded to JG from the The authors whose names are listed in the author list certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.


This work confirmed that 4-MPBA converted to 4-mercaptophenol (4-MPhOH) in the presence of H2O2. In addition, a calibration curve for H2O2 detection of 0.3 µg/mL was successfully detected in the range of 1.0 to 1000 µg/mL. Moreover, the SiO2@Au@Ag@4-MPBA for glucose detection was developed in the presence of glucose oxidase (GOx) at the optimized condition of 100 µg/mL GOx with 1-h incubation time using 20 µg/mL SiO2@Au@Ag@4-MPBA and measuring Raman signal at 67 µg/mL SiO2@Au@Ag. At the optimized condition, the calibration curve in the range of 0.5 to 8.0 mM was successfully developed with an LOD of 0.15 mM. Based on those strategies, the SERS detection of glucose can be achieved in the physiologically relevant concentration range and opened a great promise to develop a SERS-based biosensor for a variety of biomedicine applications.The importance of glucose in many biological processes continues to garner increasing research interest in the design and development of efficient biotechnology for the sensitive and selective monitoring of glucose. Here we report on a surface-enhanced Raman scattering (SERS) detection of 4-mercaptophenyl boronic acid (4-MPBA)-immobilized gold-silver core-shell assembled silica nanostructure (SiO Today, diabetes mellitus is a global problem issue with approximately 200 million people worldwide. It is of the principal causes of morbidity and disability and is largely responsible for kidney failure, heart disease, and so on . A monitHowever, a low affinity with metal surface and Raman scattering cross-section of polarizability limited the detection of glucose by SERS ,22,28,292@Au@Ag) based on the Au-seed-mediated growth of Ag on the surface of a SiO2 NP template. The presence of Au seeds facilitates and allows for the precise control of the distance and uniform of the Ag shell on SiO2 NPs as well as tuning the optical property of SiO2@Au@Ag in the visible to near IR region [2 surface was easily controlled by SiO2 core, and generating a homogenous SERS substrate of SiO2@Au@Ag [2@Au@Ag based-SERS substrates with high reproducibility and 4.2 × 106 fold-enhancement as compared to without the nanomaterial was developed for the enzyme-linked immunosorbent assay (ELISA) [2@Au@Ag NPs incubated in 4-MPBA and enzyme glucose oxidase was developed for glucose detection using SERS. The evaluation of the SERS band toward glucose level was calculated by the ratio of SERS intensity of intrinsic internal standard 4-MPBA, which was simpler than that based on the peak area of SERS bands.Recently, our group developed a gold-silver core-shell assembled silica nanostructure  ,41. Simu (ELISA) ,43,44. H3), chloroauric acid (HAuCl4), tetrakis(hydroxymethyl)phosphonium chloride (THPC), ascorbic acid (AA), polyvinylpyrrolidone (PVP), phosphate buffer saline (PBS), Tween 20, 4-mercaptophenylboronic acid (4-MPBA), 4-mercaptophenol (4-MPhOH), 4-methylbenzenethiol (4-MBT), thiophenol, 4-glucose, hydrogen peroxide (H2O2), and glucose oxidase (GOx) were purchased from Sigma–Aldrich (USA). Ethanol (EtOH) and aqueous ammonium hydroxide  were purchased from Daejung (South Korea); and ultrapure water (18.2 MΩ cm) was produced by a Millipore water purification system .Tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), silver nitrate  was briefly dispersed in 9.8 mL of water that contained 10 mg of PVP, which was kept still for 30 min. Thereafter, 20 µL of silver nitrate (10 mM) was added to the suspension, followed by the addition of 40 µL of ascorbic acid (10 mM). The suspension was incubated for 15 min to completely reduce the Ag+ ions to Ag0. By repeating the reduction steps, the final AgNO3 concentration of 300 µM was controlled. The SiO2@Au@Ag NPs were obtained by the centrifugation of the suspension at 8500 rpm for 15 min, then the NPs were washed thoroughly using EtOH to remove any excess reagent. The SiO2@Au@Ag NPs were then re-dispersed in 1 mL of absolute EtOH to obtain a 200 µg/mL SiO2@Au@Ag NP suspension.The SiO2@Au@Ag NPs, 500 µL of 2 mM 4-MPBA solution in EtOH was incubated with 500 µL EtOH contained 20 µg SiO2@Au@Ag NP for 1 h at 25 °C, followed by centrifugation for 15 min at 15,000 rpm to obtain the NP suspension. The prepared NPs were washed thoroughly using PBS containing 0.1% Tween 20 (PBST) to remove any excess reagent. The SiO2@Au@Ag@4-MPBA was then re-dispersed in 100 µL of EtOH, to obtain a 200 µg/mL SiO2@Au@Ag@4-MPBA suspension.To adsorb 4-MPBA on the surface of SiO2@Au@Ag@4-MPBA was added incubated with 100 µL of H2O2 at various concentration for 1 h at 25 °C, followed by centrifugation for 15 min at 15,000 rpm to obtain the NP suspension. The prepared NPs were washed thoroughly using PBST to remove any excess reagent. The prepared NPs was then re-dispersed in 100 µL of PBST, to obtain a 200 µg/mL NPs suspension.100 µL of PBST containing 20 µg SiO2@Au@Ag@4-MPBA was added, incubated with 100 µL PBST that contained various concentrations of glucose, and 100 µL of GOx for 1 h at 25 °C, followed by centrifugation for 15 min at 15,000 rpm to obtain the NP suspension. The prepared NPs were washed thoroughly using PBST to remove any excess reagent. The prepared NPs was then re-dispersed in 100 µL of PBST, to obtain a 200 µg/mL NPs suspension.One hundred microliters of PBST containing 20 µg SiO−1.To obtain the surface-enhanced Raman spectra, a micro-Raman system with a 10-mW 532-nm diode-pumped solid-state laser excitation source and an optical microscope  was utilized. The SERS signals were collected with a back-scattering geometry using an objective lens with a magnification of 10× . The selected sites were measured randomly, and all the SERS spectra were integrated over a period of 5 s. The size of the laser beam spot was approximately 2.1 μm, and the SERS spectrum was obtained within the wavenumber range of 300–1700 cm2@Au@Ag was first immobilized by 4-MPBA as a Raman reporter and labeling molecule. Then this suspension was added into glucose solution in the presence of the GOx enzyme. GOx enzyme catalyzed and converted glucose to gluconolactone and H2O2. The presence of H2O2 converted 4-MPBA to 4-MPheOH on the surface of SiO2@Au@Au. The variation of SERS signal of 4-MPBA was observed and extrapolated to the concentration of glucose in solution  were first functionalized by 3-aminopropyltriethoxysilane (APTS) to prepare aminated silica NPs. Simultaneously, colloidal Au NPs (3 nm) were synthesized by THPC and incubated with the aminated silica NPs by to prepare Au NPs seed embedded with SiO2 (SiO2@Au seed NPs), according to the method reported by Pham et al. [2@Au seed were grown by reducing a silver precursor (AgNO3) in the presence of ascorbic acid (AA) and polyvinylpyrrolidone (PVP) [2@Au seed to generate the SiO2@Au@Ag nanostructure. According to a previous study, 4-MPBA was used for specific binding of glucose with the boronic acid motif in 4-MPBA, leading to a significant increase in the absolute intensity of the SERS signal of 4-MPBA. So, we modified the surface of SiO2@Au@Ag by 4-MBPA to generate SiO2@Au@Au@4-MPBA as a specific ligand for glucose. Subsequently, glucose was added in the reaction in the presence of glucose oxidase (GOx). GO catalyzed and converted glucose to glucolactone and hydrogen peroxide (H2O2) in the reaction. H2O2 converts 4-MPBA to 4-mercaptophenol (4-MPhOH).In this study, SiOsolution . To prepm et al. ,42,43,44ne (PVP) . The sil2@Au@Ag NPs are first shown in 2@Au and SiO2@Au@Ag NPs are shown in 2 NPs when the colloidal Au NPs were incubated with the amine-functionalized silica NPs for 12 h at 25 °C  and with 100 µM 4-MPBA, we found that the SiO2@Au@Ag in EtOH solution showed SERS bands at 432, 883, 1051, 1097, 1276, and 1455 cm−1, assigned to the SERS bands of the EtOH solution. At 100 µM 4-MPBA, clear and new bands were observed at 417, 473, 1077, 1170, 1484, and 1583 cm−1, indicating that 4-MPBA was successfully immobilized on the surface of SiO2@Au@Ag [2@Au@Ag@4-MPBA can be seen in 2@Au@Ag@4-MBPA was also examined in the presence of various concentrations of 4-MPBA from 0.1 µM to 100 µM in EtOH solution. The SERS signal of 4-MBPA increased from 0.1 to 10 µM and achieved a saturation at 50 µM. In particular, the intensities of the Raman bands at 1583 cm−1 increased dramatically with 4-MPBA concentration .As mentioned above, we chose the SERS band of 4-MPBA at 417, 1077, and 1583 cmiophenol ,46,47. Tnol [2O2 . The SER597 cm−1 a. Based 597/1583 . The dra2@Au@Ag@4-MPBA in the presence of GOx were examined and optimized in Next, the effect of experimental conditions for glucose detection on the SERS signal of SiO2@Au@Ag@4-MPBA for 30 min while the GOx concentration was varied in the range of 10−2–103 µg/mL. The results showed that the SERS signal of the SiO2@Au@Ag@4-MPBA at 5 mM glucose and various concentration of GOx in both the “step-by-step” and “all-in-one” techniques were quite similar . The theoretical LOD was 0.15 mM, estimated by the 3sblank criterion. Our result demonstrates that our material can be utilized for glucose detection for diagnosis.A linear curve-fitting procedure was utilized for calibration. A significant relationship between the SERS signal ratio and the glucose concentration was found in the experimental data points ranging from 1.0 to 8.0 mM , uric acid (UA), and bovine serum albumin (BSA), at various concentrations and fructose co-exist with glucose in blood samples ,49,50. Fg@4-MPBA . The SER2@Au@Ag@4-MPBA was examined in 2@Au@Ag@4-MPBA was synthesized, re-dispersed in EtOH, and stored at 4 °C for one week. The SERS signal of 4-MPBA was measured every day and the SERS signal at 1583 cm−1 was monitored. As showed in 2@Au@Ag was not oxidized during storage in EtOH at 4 °C. However, the SERS signal slightly increased after four days, which might be from the partly aggregation of SiO2@Au@Ag.The long-term storage of the SiO2@Au@Ag@4-MPBA for quantitative, selective detection of glucose in neutral condition. This work confirmed that 4-MPBA was converted to 4-MPhOH in the presence of H2O2. Moreover, it provided a new calibration curve to evaluate H2O2 species in the range of 1.0 to 1000 µg/mL with LOD as low as 0.3 µg/mL. Moreover, the SiO2@Au@Ag@4-MPBA for glucose detection in the presence of GOx were optimized at 100 µg/mL GOx, 100 µg/mL GOx with 1-h incubation time using 20 µg/mL SiO2@Au@Ag@4-MPBA and measuring Raman signal at 67 µg/mL SiO2@Au@Ag. At the optimized condition, the calibration curve for selective glucose detection in the range of 0.5 to 8.0 mM was successfully developed with an LOD of 0.15 mM. The combination of GOx and our nanostructure also illustrated that our SERS probe can be coupled with other enzymes to greatly expand its applicability to biologically active targets.We have developed a new SERS-based boronated nanoprobe of the SiO


The significant reduction of new antibiotics development can be overcome by complementing with alternative therapies, such as antimicrobial photodynamic therapy (aPDI). Through photosensitizer (PS) compounds, aPDI produces local oxidative stress-activated by light (photooxidative stress), nonspecifically killing bacteria. Methodology: Bimetallic Re(I)-based compounds, PSRe-µL1 and PSRe-µL2, were tested in aPDI and compared with a Ru(II)-based PS positive control. The ability of PSRe-µL1 and PSRe-µL2 to inhibit K. pneumoniae was evaluated under a photon flux of 17 µW/cm2. In addition, an improved aPDI effect with imipenem on KPC+ bacteria and a synergistic effect with cefotaxime on ESBL producers of a collection of 118 clinical isolates of K. pneumoniae was determined. Furthermore, trypan blue exclusion assays determined the PS cytotoxicity on mammalian cells. Results: At a minimum dose of 4 µg/mL, both the PSRe-µL1 and PSRe-µL2 significantly inhibited in 3log10 (>99.9%) the bacterial growth and showed a lethality of 60 and 30 min of light exposure, respectively. Furthermore, they were active on clinical isolates of K. pneumoniae at 3–6 log10. Additionally, a remarkably increased effectiveness of aPDI was observed over KPC+ bacteria when mixed with imipenem, and a synergistic effect from 3 to 6log10 over ESBL producers of K. pneumoniae clinic isolates when mixed with cefotaxime was determined for both PSs. Furthermore, the compounds show no dark toxicity and low light-dependent toxicity in vitro to mammalian HEp-2 and HEK293 cells. Conclusion: Compounds PSRe-µL1 and PSRe-µL2 produce an effective and synergistic aPDI effect on KPC+, ESBL, and clinical isolates of K. pneumoniae and have low cytotoxicity in mammalian cells.Background: Extended-spectrum beta-lactamase (ESBL) and carbapenemase (KPC Klebsiella pneumoniae producing extended-spectrum β-lactamase (ESBL) and carbapenemase (KPC) are among the most relevant   ,7 phenan  ,7 phenan  . The phoroducers . In addi3Cl)2µ-N^N, where N^N is 2,3-Dicarboxypyrazino   ,7 phenan  ,7 phenan+) of K. pneumoniae were used for the antimicrobial assay. Additionally, 118 clinical isolates of K. pneumoniae were used. Those clinical isolates were previously characterized for sensitivity and pathogenicity patterns (PF6)2  compound was used as a control to compare the aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds [−1cm−1 [The PS-Ru [Ru(bpy)ompounds . Accordiompounds  and a si [−1cm−1 . In addiK. pneumoniae. PSRe-µL1 and PSRe-µL2 at 16 µg/mL were then mixed with each K. pneumoniae strain, KPPR1 (KPC−) and ST258 (KPC+) at 1 × 107 CFU/ml. The aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds was compared with the reference PS-Ru antimicrobial activity as a positive control [10 (<99.9%) the growth of both strains of K. pneumoniae (* p < 0.05) compared to untreated control bacteria (Blue bars). The results show that the bactericidal effect produced by both PSs is dependent on light (Red bars)  because growth inhibition is observed after light activation. Therefore, the PSRe-µL1 and the PSRe-µL2 compounds must be activated by light to exhibit their bactericidal effect. Comparable results were obtained with the 16 µg/mL PS-Ru control, as the bacterial growth inhibition was observed only after light activation (p < 0.05).The photodynamic antimicrobial capacity of the two new Re (I) compounds was determined by inhibiting the bacterial growth of  control ,38,49. IK. pneumoniae strains, a further two pharmacologic parameters were determined: the minimum effective concentration (MEC) and the minimum light exposition time  on the KPPR1 and the ST258 K. pneumoniae strains. The PS-MEC was determined by exposition of 1 × 107 CFU/mL of each K. pneumoniae strain to serial dilutions (ranging from 0 to 32 µg/mL) of each PS in ca-MH broth. The aPDI treatment was performed for 1 h at a fluence rate of 17 µW/cm2. After treatment, viable bacteria were enumerated as above by serial micro-dilution. We established the MEC as the concentration where the bacterial viability decreased in 3 log10 (99.9%). As shown in p < 0.01, *** p < 0.001, Student’s t-test comparing to control with 0 µg/mL). The MEC of 4 µg/mL of PSRe-µL1 or PSRe-µL2 were used to determine the lethality time on 1 × 107 CFU/mL of each bacterial strain in ca-MH broth. The mix was exposed to 17 µW/cm2 of a white LED light for 5, 15, 30, 60, and 120 min. Control wells with bacteria without PS were also included. For each time, viable bacteria were enumerated by serial micro-dilution and colony counted in ca-MH agar. We established the lethality when the bacterial viability decreased in 3 log10 (99.9%). As seen in p = 0.013, compared to time 0), a 3log10 reduction was observed after 60 min of light exposure . In comparison, the PSRe-µL2 significantly (p < 0.01) reduced in 3log10 the bacterial viability after 30 min (Because the PSRe-µL1 and PSRe-µL2 compounds showed a significant aPDI effect on the two r 30 min D.K. pneumoniae. The strains of K. pneumoniae susceptible to carbapenem KPC− (KPPR1), and the resistant strain KPC+ (ST258), were exposed to the preparation of 4 µg/mL of imipenem mixed in aqueous solution with 4 µg/mL of each PSRe-µL1, PSRe-µL2 (corresponding to their MECs), or the control compound PS-Ru [p < 0.001) increased effect over bacterial viability, increasing from 3 to 6log10 the bactericidal effect for the KPC+ strain (10 inhibitory effect (p < 0.05) [The desired quality for photosensitizer compounds is to be used as adjunctive therapy with antibiotics. The improvement in combined therapy of the compounds PSRe-µL1 and PSRe-µL2 with imipenem was used to verify their usefulness in eradicating carbapenemase-producing nd PS-Ru . Additio+ strain . As seen < 0.05) .K. pneumoniae from patients who had an active infection [p < 0.001; compared to untreated control) inhibits bacterial growth > 3log10 (>99.9%) of clinical isolates of K. pneumoniae. The results show that the bactericidal effect produced by PSRe-µL1 and PSRe-µL2 is light-dependent . Those results are comparable with the positive control PS-Ru (4 µg/mL) (*** p < 0.001). Because this collection of strains has been characterized by its resistance profile and presents 66 ESBL-producing isolates, we analyzed whether a synergic effect occurred with cefotaxime. It was first determined whether the combined treatment with Cfx increases the inhibition of bacterial growth of aPDI with PSRe-µL1 or PSRe-µL2. The clinical isolates were exposed to the preparation of 4 µg/mL of cefotaxime with 4 µg/mL of PSRe-µL1 or PSRe-µL2. As expected, the PSs compounds mixed with cefotaxime significantly (*** p < 0.001) increased the bactericidal effect on the clinical isolate population from 3 to 6log10 reduction (p > 0.05).We have in our laboratory a collection of 118 clinical isolates of nfection . We usednfection . As seeneduction . No signK. pneumoniae [p < 0.001) from 8 µg/mL (8–8) to 0.17 µg/mL (0.09–0.34) with PSRe-µL1 and 0.23 µg/mL (0.15–0.41) with PSRe-µL2 on Cfx-MIC was observed  index was determined to verify when the PS and Cfx combination increases the bactericidal activity synergistically or additively. We used the MEC determination for the photosensitizer compounds, and the results of the mixture with antibiotics were tabulated as the MIC for simplicity. The set of 66 ESBL-producing clinical isolates of eumoniae  were mixobserved . In addiobserved . Both PSobserved . Figure 2 of white LED light when the PSs were removed. Fresh medium DMEM with 10% FBS without PS was replaced, and cells were incubated 24 h more in the dark at 37 °C in a 5% CO2 atmosphere. As shown in p > 0.05, Student’s t-test, comparing treated cells with the control). Similarly, the light exposition induced no significant cell death for HEp-2 cells, although a slight (12.5 ± 5%) but significant (p > 0.05) reduction in cell viability of PSRe-µL1 over HEK293T cells were shown.The PSRe-µL1 and PSRe-µL2 compounds were tested in vitro and found to be secure for mammalian cells. This work tested the intrinsic cytotoxicity (dark cytotoxicity) and light-dependent cytotoxicity of the PSs compounds in the human HEp-2 and HEK293 cell lines. The trypan blue exclusion technique allowed us to determine cell death of 500,000 cells in the presence of 4 µg/mL PSRe-µL1 or PSRe-µL2. The cells were incubated with PSs for 1 h in the dark or activated with 17 µW/cmK. pneumoniae [Previously, we reported photosensitizing compounds based on Ir (III), which exhibited absorption processes close to 400 nm wavelength . Therefoeumoniae ,3. In theumoniae .K. pneumoniae cell envelope. Additionally, by exciting these high-rate light-absorbing dark compounds, they can produce high-energy triplet states, which promote energy transfer to molecular oxygen [3MLCT, 3LC, or a mixture of both states [The effective microbial activity of the PSRe-µL1 and PSRe-µL2 photosensitizers rests on the photophysical properties provided by their chemical structure. Although these compounds are neutral (do not have positive charges in the coordination sphere), they can easily polarize by absorption of light, as described in a previous report . This chr oxygen . At firsr oxygen . It willh states . Although states . A more K. pneumoniae (>3log10). These results are comparable to using cyclometalated Ir (III) complexes as aPDI, as previously reported [Acinetobacter baumannii [K. pneumoniae [K. pneumoniae. We used this collection of strains because its antibiotic resistance is characterized and presents a well-established sub-population of ESBL-producing bacteria [At concentrations as low as 4 µg/mL, both the PSRe-µL1 and the PSRe-µL2 compounds showed an effective aPDI activity, inhibiting the growth of reported ,29. Simireported ,54,55,56aumannii . Similaraumannii . This beeumoniae . Howeverbacteria . The aPDbacteria ,57. SimiThe antimicrobial activity of the PSRe-µL1 and PSRe-µL2 is photodynamic and, therefore, its activation is dependent on light. The dependence on light suggests that the PS compounds themselves and in the dark are not toxic, as the results with mammalian cells show us. The slight but significant light-dependent cytotoxicity exhibited by PSRe-µL1 on HEK293T cells reinforces the low intrinsic toxicity. However, it may imply potential damage to the host tissues that must be considered and prevented with a better characterization of the exposure times and concentration of the compound. Furthermore, the cytotoxicity shown by the PSRe-µL1 and PSRe-µL2 compounds is of low significance compared to that shown by the antitumor photosensitizer compounds . However2 of white light, they are bactericidal. Compounds with optimum absorbance at higher wavelengths ranging from 450–750 nm would improve the exposure of PS to light into the tissues, but being less energetic, the PSs must have a triplet excited state of easy access to promote energy transfer [For now, it is difficult to accurately calculate the dose of light necessary to activate these PSRe compounds fully; however, we observed in vitro that with photon flux as low as 17µW/cmtransfer . The dartransfer . Therefotransfer .The present study shows that the increased MICs of resistant bacteria could be reverted by aPDI, turning resistant strains into susceptible ones. Thus, aPDI would effectively treat MDR bacteria, greatly complementing antibiotic therapy. Furthermore, therapeutic regimens with lower doses of antibiotics and PS can be used effectively due to the synergistic effect. The requirement for lower doses of antibiotics will help reduce the generation of resistance. In this work, two bimetallic Re(I) compounds were tested as PSs to be used in the aPDI. According to their UV-vis absorption characterization, it was identified that the absorptions at lower energies occur at wavelengths higher than 450 nm, improving its use in infections compared to the PSs based on Ir(III), previously studied. The dark quality in both powder solids and liquid solutions of the Re(I) compounds may imply that light of a wide wavelength range can be absorbed , increas