In vitro results show that under 808 nm near-infrared irradiation, killing effect of the nanoprobes on drug-resistant MIA PaCa-2 cell is 3.3 times than that of GEM alone. In vivo experiments indicate the synergetic photothermal-chemotherapy not only loosens fibrous matrix of pancreatic tumor model, but also dramatically inhibits tumor growth, and almost completely eradicates the tumor after 12 days of treatment. In addition, relaxation rate of the nanoprobes is 8.2 times than commercial contrast agent Magnevist, therefore boosts the signal of magnetic resonance imaging in pancreatic tumor. In conclusion, our results reinforce that the prepared nanoprobes are promising to break matrix barrier and overcome drug resistance in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignant tumors with features of matrix barrier caused poor drug permeability, and susceptibility to drug resistance. Herein, a PDAC and its stromal cell dual-targeted photothermal-chemotherapy strategy is explored to loosen the matrix and reverse drug resistance. To achieve this goal, black TiOThe online version contains supplementary material available at 10.1186/s12951-022-01525-3. Pancreatic ductal adenocarcinoma (PDAC) is a severely malignant tumor, with a poor 5-year survival rate of only 9%, and it is estimated to be the second largest contributor to cancer-related death after lung cancer by 2030 –3. Chemo3 and WS2, have been reported to destroy the dense matrix barrier of pancreatic cancer and enhance the chemosensitivity of drug-resistant cells, and therefore can eradicate drug-resistant PDAC cells  carbodiimide hydrochloride (EDC), Gadolinium nitrate hexahydrate (GdN3O9·6H2O), N-hydroxysuccinimide (NHS), Rhodamine 123 (C21H17ClN2O3) were obtained from Macklin Biochemical Co., Ltd . Carboxyl polyethylene glycol amino group (NH2-PEG-COOH) were obtained from Yare Bio Co., Ltd . Recombinant human IGF1 protein (ab270062) was purchased from Abcam Bio Co., Ltd (England). Gemcitabine (C9H11F2N3O4) was purchased from MedChemExpress Co., Ltd (USA). DMEM medium, 0.25% Trypsin–EDTA was acquired from Gibco . Penicillin–streptomycin liquid, phosphate buffer saline (PBS) came from GE Healthcare HyClone . 4% formaldehyde solution, Phalloidin, Fluorescein Isothiocyanate Labeled (FITC), Hoechst 33258 (C25H24N6O·3HCl), RIPA buffer were obtained from Solarbio Biotech Co., Ltd . The calcein-AM/PI double stain kit came from Yeasen Biotechnology Co., Ltd . Protease inhibitor Cocktail was purchased from Bimake Biotechnology Co., Ltd (USA). ECL chromogenic solution was purchased from Adavansta Co., Ltd (USA). PVDF membrane was purchased from Millipore Co., Ltd (USA). Skim milk powder was purchased from Yili Co., Ltd . Pre-stained protein MARKER (BIO-RAD) was purchased from Thermo Fisher Scientific Inc (USA). BCA Protein Assay Kit was obtained from Beyotime Biotech Co., Ltd . β-Actin Mouse Monoclonal Antibody was purchased from Santa Cruz Biotech Co., Ltd (USA). Anti-IGF1R antibody was acquired from Bioss Biotech Co., Ltd .Dopamine hydrochloride, sodium borohydride  and were dispersed via ultrasound. Then ultrapure water was introduced to the bTiO2 solution to 90 mL. 500 mg of PAA2000 powders were dissolved into 10 mL of ultrapure water and were stirred magnetically. Next, the bTiO2 mixture was combined with PAA2000 and stirred for 5 h at room temperature (RT). The dispersions were next collected and centrifuged at 12,000 rpm for thrice. The bTiO2-PAA nanoparticles were dissolved in 90 mL of ultrapure water. 10 mL of Gadolinium Acetate Tetrahydrate (1 mg mL−1) were added in bTiO2-PAA dispersions. After stirring for 3 h, the dispersions were collected and centrifuged at 12 000 rpm thrice, then obtained bTiO2-Gd were dispersed in 10 mL of ultrapure water. The bTiO2-Gd dispersions were added in 80 mL of 10 mM Tris (PH8.5). After ultrasound for 10 min, the 2 mL of dopamine hydrochloride (75 mg mL−1) was introduced dropwise into this reaction system and stirring at RT for 1 h. The bTiO2-Gd-PDA dispersions were collected by centrifugation. Subsequently, the dispersions were dispersed into 50 mL of ultrapure water. After ultrasound for 10 min, NH2-PEG-COOH solutions were added dropwise to the bTiO2-Gd-PDA and stirring for 24 h. Finally, the bTiO2-Gd-COOH dispersions were centrifuged at 12,000 rpm, prior to storage in 10 mL of ultrapure water.Gadolinium acetate tetrahydrate, dopamine hydrochloride and NH2-Gd-COOH nanoparticles were conjugated with IGF1 peptide by EDC/NHS activated amide bonds. Briefly, 5 mL of bTiO2-Gd-COOH dispersions were resuspended in 45 mL of ultrapure water. 15 mg EDC and 20 mg NHS were dissolved in the dispersions, followed by stirring at RT for 20 min. Subsequently, 100 μL of IGF1 peptide (1 μg μL−1) were added and stirred for 12 h. The prepared bTiO2-Gd-IGF1 was collected by centrifugation and stored at 4 degrees.bTiO−1) were introduced to 8 mL of bTiO2-Gd-COOH (0.75 mg mL−1) and bTiO2-Gd-IGF1 (0.75 mg mL−1) respectively, followed by stirring for 24 h. GEM loaded nanocarriers (bTiO2-Gd-GEM or bTiO2-Gd-IGF1-GEM), the nanoparticles were centrifuged, followed by storage in ultrapure water. The GEM loading efficiency was computed based on the drug’s UV–visible absorption, as we reported earlier [−1) UV–visible absorptions, as well as the GEM concentration–absorption curve. Lastly, the GEM loading amount on the nanocarriers was computed as follows: loading efficiency =  ÷ nanocarrier amount) × 100%.2 mL of GEM . The nanoprobe zeta potential and nanoprobe size distribution were next examined via a particle-size zeta potential analyzer , and by implementing a UV–visible spectrophotometer , UV–visible absorption spectra were assessed. Infrared (IR) spectra of the nanoprobes were obtained from a Fourier transform infrared spectrometer . The nanoprobe element content was measured via induction coupled plasma optical emission spectrometry . Further, by employing a MR scanner system utilizing a magnetic field of 1.5 T, MR imaging and relaxivity capabilities were tested. The nanoprobe photothermal stability and effectiveness were assessed via an infrared (IR) thermal imaging apparatus  and 808 nm semiconductor laser .2-Gd-COOH nanoparticles with different Ti concentrations were added to cuvette and irradiated with various power densities from an 808 nm laser. The values of temperature for bTiO2-Gd-COOH dispersions were documented every 10 s using an IR thermal imaging apparatus , and we simultaneously mapped the images of real-time thermal dispersion. We have summarized the different aforementioned situations below: (1) 1 mL of nanoparticles (150 μg mL−1 of Ti concentration) receiving irradiation with 808 nm laser with different power densities (0.4–2.0 W cm−2) for 10 min; (2) Varying nanoparticle concentrations (0–150 μg mL−1 of Ti concentration) were irradiated through an 808 nm laser (1.2 W cm−2) for 10 min; (3) For detecting nanoparticle photostability, the bTiO2-Gd-COOH dispersions (150 μg mL−1 of Ti concentration) were also irradiated with an 808 nm laser (1.2 W cm−2) every 5 min for over 6 on/off repeating cycles. The resulting images were collected, and the temperature values fitted in time–temperature curves.To test photothermal properties, bTiO2-Gd-IGF1-GEM (GEM: 3 μg mL−1) was placed, and each dialysis bag was immersed in 20 mL PBS buffer solution with pH of 7.4, 5.0 and 5.0 respectively. 1 mL of dialysate was withdrawn and the same quantity of PBS buffer was introduced to keep the same quantity of buffer equal at 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, 12 h, 24 h, and 27 h, respectively. Upon collection of the solution, the solution with pH 5.0 received irradiation with a 1.2 W cm−2 near-infrared laser for 15 min. The drug release was monitored via UV–Vis spectrophotometer, and the drug release was computed based on the drug release dose absorption curve.To evaluate drug release, in three dialysis bags, 2 mL bTiO1) and (r2) relaxivities from the rate of relaxation R1 (1/T1) and R2 (1/T2) slopes accordingly. The T1-weighted MRI characteristics of nanoparticles at varying concentrations of Gd were obtained from the same MRI apparatus, and parameters were adjusted as shown below: T1 spin echo sequence, TE = 18.20 ms, TR = 200.00 ms, FOV = 100 mm × 100 mm, slice width = 2.0 mm, 1.5 T.The MRI and MR relaxivity of nanoprobes were evaluated by 1.5 T MRI scanner apparatus. The nanoprobes with varying Gd concentrations  were placed in a tube to measure the relaxation rate. At the same time, the commercial contrast agent Magnevist was adjusted to the control Gd concentration. We obtained the specific longitudinal and transverse, namely . The MIA PaCa-2, Panc-2, BxPC-3 and hTERT-HPNE cell lines were grown in complete DMEM medium, containing 10% FBS, 100 U mL2-Gd-COOH and bTiO2-Gd-IGF1, MIA PaCa-2 PDAC cells were seeded in 96-well plates (1 × 104 cells per well) and grown for 24 h. Next, different concentrations of nanoprobes (50–300 μg mL−1) were added into each well. Following 20 h, 10 μL of MTT reagent (5 mg mL−1 in PBS) was introduced to each well and kept for additional 4 h. Finally, the mediums were suctioned off, and formazan crystals were resuspended by DMSO. The absorbances of formazan solutions were detected using a microplate absorbance reader  at 490 nm and 550 nm, and cell viabilities were computed.For cytotoxicity analysis of bTiO2-Gd-COOH, bTiO2-Gd-IGF1, bTiO2-Gd-IGF1-GEM for 2 h, respectively. Next, cells were twice PBS-rinsed before fixation in 4% formaldehyde solution. Finally, elemental fluorescence of Ti and Cl in cells was obtained. The X-ray energy was 10 keV, and the beam spot was 0.5 × 0.5 μm2. Elemental maps of Ti, Cl in cells were acquire.To measure cell-targeting capability of IGF1 conjugated nanoprobes, X-ray fluorescence microscopy at Shanghai Synchrotron Radiation Facility  was used. First, cells were seeded on sterile Malay films and placed in a cell incubator for 24 h, then cultured in DMEM and bTiO2-Gd-COOH, bTiO2-Gd-IGF1 for another 2 h. Next, the cells were digested by nitric acid and hydrofluoric acid. The element content in cells were determined via ICP-OES.For statistical analysis of targeting ability, MIA PaCa-2 cells were seeded in 6-well plates for 24 h, and maintained with bTiO2-Gd-COOH and bTiO2-Gd-IGF1 nanoprobes were labelled by fluorescence dye rhodamine 123, and were determined by laser confocal microscope at excitation wavelength of 488–505 nm and emission wavelength of 515–575 nm. MIA PaCa-2 cells (1 × 105 cells) were grown on a 35 mm petri dish and placed in an incubator for 24 h, prior to treatment with the same concentration of DMEM, bTiO2-Gd-COOH, and bTiO2-Gd-IGF1 for 2 h. Next, the cells were PBS-rinsed before fixation in 4% paraformaldehyde, and treatment with 0.2% Triton X-100. Followed, 1% BSA solution was used for blocking non-specific interacting sites, and the cells were incubated by employing 50 μg mL−1 FITC-Ghost Pencyclic peptide and 2 μg mL−1 Hoechst 33342 for cytoskeleton and nucleus staining.For furthermore proving targeting capability, bTiO4 cells per well), followed by incubation in DMEM, GEM, bTiO2-Gd-COOH, bTiO2-Gd-GEM, and bTiO2-Gd-IGF1-GEM or bTiO2-Gd-IGF1 for 2 h. Ti concentrations of bTiO2 contained groups were 150 μg mL−1, the GEM concentration of the GEM alone group was consistent with that of the other groups, which was 3 μg mL−1. 2 h later, the old media was suctioned off and fresh DMEM was added, and then each well underwent irradiation with 808 nm NIR laser for 1–4 min at power densities of 1.2 W cm−2, respectively. MTT was subsequently employed to assess cell viability.MIA PaCa-2 cells were grown in the plates containing 96 wells  staining.For animal experiments, animal care and handling procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments at Ningbo University (Permit No. SYXK Zhe 2019-0005). To assess nanoparticle toxicity in vivo, 12 normal Balb/c mice (4–6 weeks old) were arbitrarily separated into 3 groups. The mice received intravenous injection of 150 μL of PBS, bTiO6 cells for each mouse) in DMEM were subcutaneously injected in the right flank of the animals. The mice were arbitrarily separated into 6 groups (5 mice per group). The tumor size was measured with a digital caliper. Tumor volume = 4/3 × π × (tumor radius)3. Our experiments were initiated once tumors reached 50 mm3. The mice received intratumoral administration of 100 μL of PBS, GEM, bTiO2-Gd-IGF1, bTiO2-Gd-IGF1-GEM. The concentration of GEM was 3 μg mL−1 in GEM groups and the concentration of Ti concentration was 150 μg mL−1 in the bTiO2 groups. Meanwhile, for MR imaging, commercial contrast agent magnevist with a Gd concentration of 3 μg mL−1 was injected. Following 24 h of incubation, mice underwent intraperitoneal injection of 8 wt% of chloral hydrate solution, followed by irradiation, in the presence or absence of 808 nm NIR laser for 5 min at 1.2 W cm−2. The tumor temperature alterations were documented using an infrared thermometer . From each group, one mouse was arbitrarily chosen and euthanized following the irradiation of NIR, and the tumor injury was analyzed via H&E staining. We monitored tumor sizes for 12 days and computed the volumes based on the aforementioned tumor volume formula. Mice body weights were also documented for 2 weeks.Tumor models were developed in male balb/c nude mice (4–6 weeks). MIA PaCa-2 cells , with phenolic hydroxyl polymerized special cyclic structures, can supply binding sites for peptide conjugation and drug loading, and is usually used for surface coating of nanoparticles [2-Gd-PDA, the nanoprobes were conjugated with amino group of NH2-PEG-COOH through Michael addition reaction. The formed bTiO2-Gd-COOH nanoprobes have good dispersibility. Gemcitabine (GEM), a first-line chemotherapy drug for PDAC, was loaded by bTiO2-Gd-COOH via pai–pai stacking and/or hydrogen binding [2-Gd-IGF1 nanoprobes [The bTiOarticles , 37. To  binding . Finallynoprobes .2-Gd, bTiO2-Gd-PDA and bTiO2-Gd-IGF1 nanoprobes are 60 nm, from left to right respectively. The mean hydrated size of bTiO2-Gd, bTiO2-Gd-PDA, bTiO2-Gd-COOH, bTiO2-Gd-IGF1, bTiO2-Gd-IGF1-GEM are about 123.3 nm, 153.2 nm, 149.2 nm, 158.8 nm and 159.9 nm, respectively, in Fig. 2-Gd-IGF1-GEM of this size has good biocompatibility and is still suitable for tumor treatment. In addition, zeta potentials of bTiO2-Gd, bTiO2-Gd-PDA, bTiO2-Gd-COOH, IGF1 polypeptide, bTiO2-Gd-IGF1 and bTiO2-Gd-IGF1-GEM are about 24.73 mV, − 4.85 mV, − 17.87 mV, 0.67 mV, − 15.27 mV and − 25.67 mV, respectively. In general, traditional white TiO2 nanoparticles show negative zeta potential owing to surface hydroxyl groups. Our previous investigation prove bTiO2 nanoparticles exhibit positive zeta potential which probably due to reduction induced abundant oxygen vacancies on the nanoparticles surface [3+ also increase positive charge, so the surface charge of bTiO2-Gd nanoprobes appear positive charge. After coating with PDA, surface charge of bTiO2-Gd-PDA nanoprobes are changed to negative. Zeta potential of bTiO2-Gd-IGF1-GEM reaches − 25.67 mV after modification with NH2-PEG-COOH, IGF1 and GEM, which is more suitable for in vivo delivery when compared with positive surface charge.As shown in Fig.  surface . Besides−1 in bTiO2-Gd-PDA, bTiO2-Gd-COOH, and bTiO2-Gd-IGF1, which is attributed to characteristic absorption peak of Ti–O [2-PEG-COOH through Michael addition reaction, bTiO2-Gd-COOH nanoprobes exhibit carboxylic acid symmetrical and asymmetrical stretching vibrations at 1386 cm−1 and 1573 cm−1 [2-Gd-IGF1 nanoprobes, there are characteristic peaks at 1494 cm−1, 1153 cm−1, and 1211 cm−1, which suggests IGF1 polypeptide has been successful connected with bTiO2-Gd-COOH nanoprobes. UV–visible spectra of various samples are provided in Fig. 2-Gd-COOH and bTiO2-Gd-IGF1, the absorption peak of the bTiO2-Gd-IGF1-GEM nanoprobes exhibit slightly red-shift which attributing to gemcitabine loading.Figure  of Ti–O . After m573 cm−1 . In bTiO1 value of bTiO2-Gd-IGF1 is 38.496 mM−1 s−1, while the value of Magnevist is 4.7248 mM−1 s−1 at the similar concentration of Gd. It should be pointed out that the bTiO2-Gd-IGF1 r1 value is approximately 8.2 folds greater than the Magnevist. The markedly high bTiO2-Gd-IGF1 r1 value is likely because of gadolinium coordinated by PAA with hydrophilic high molecular weight, which can enhance the rotational correlation time (τR) [2 values of bTiO2-Gd-IGF1 is also markedly elevated (r2 = 57.2529 mM−1 s−1), compared to Magnevist (r2 = 4.6498 mM−1 s−1). The bTiO2-Gd-IGF1 r2/r1 ratio is approximately 1.487, which illustrates the nanoprobes are good T1-weighted MRI contrast agent. Consequently, it is clearly indicated bTiO2-Gd-IGF1 nanoprobes exhibit brighter T1-weighted MRI signal than Magnevist at the same Gd concentration in Fig. MR imaging and relaxivities of the nanoprobes were evaluated by 1.5 T MRI scanner system. Commercial contrast agent Magnevist is employed as the control. As depicted in Fig. ime (τR) . Similar2 nanoparticles possess high photothermal capability [2 nanoparticles were modified by PAA, PDA, as well as NH2-PEG-COOH, therefore, photothermal performance of the prepared bTiO2-Gd-COOH nanoprobes were evaluated. Figure 2-Gd-COOH dispersions (150 μg mL−1) were irradiated with 808 nm laser for 10 min. With 808 nm laser increase in power, the nanoprobe temperature rises quickly after 10 min. As expected, cancer cells can undergo rapid ablation following few minutes of exposure to 50 °C [−2, the temperature of bTiO2-Gd-COOH dispersions were 62.2 °C after 10 min irradiation, which is considered more suitable in followed experiment. Then, bTiO2-Gd-COOH nanoprobes underwent irradiation with 1.2 W cm−2 of NIR laser at various concentrations of Ti from 50 to 150 μg mL−1. As shown in Fig. −1 of bTiO2-Gd-COOH nanoprobes reached 48.7 °C after irradiated by 808 nm laser for 10 min. In Fig. 2-Gd-COOH nanoprobes were irradiated by an 808 nm laser at 1.2 W cm−2 for 6 cycles in 1 h. The temperature curve during heating and cooling is almost similar during in 6 cycles, which proves nanoprobes are stability during NIR irradiation.Although previous reports have proved bTiOpability , 29. In to 50 °C . When th2-Gd-IGF1-GEM is about 10.3%, which indicates high loading capacity of bTiO2-Gd-IGF1 nanoprobes. Additional file 2-Gd-IGF1-GEM at pH 7.4, pH 5.0, pH 5.0 + NIR, respectively. The cumulative release of GEM within 27 h was 2.7 times higher in the pH 5.0 group, compared to the pH 7.4 group. The weak acid promoted drug release is probably due to the protonation of amine groups on the PDA scaffolds and/or on the GEM molecules, which partially weeks the pai–pai interactions between PDA and drugs [2-Gd-IGF1-GEM nanoprobe exhibit pH and NIR co-responded drug-release properties, consequently can enhance the concentration of GEM in the tumor's weakly acidic microenvironment.According to the above-mentioned formula, drug loading efficiency of bTiOnd drugs . After nnd drugs , 43. TheWestern blot was employed to quantitatively determine the expression level of IGF1R in several PDAC cell lines, such as MIA PaCa-2 cells, hTERT-HPNE cells, BxPC-3 cells, and Panc-2 cells. Additional file 2-Gd-IGF1 nanoprobes were generated via conjugation of bTiO2-Gd-COOH with IGF1 peptides. The bTiO2-Gd-IGF1 nanoprobe targeting ability was evaluated by XFM, ICP-OES and laser confocal microscope. By taking advantage of XFM, direct visual and quantitative localization of the titanium element was acquired. As depicted in Fig. 2-Gd-COOH group, suggesting that MIA PaCa-2 cells have weak uptake of bTiO2-Gd-COOH nanoprobes. However, intensive yellow fluorescence appears in bTiO2-Gd-IGF1 and bTiO2-Gd-IGF1-GEM groups, which directly suggests that IGF1 peptide can enhance the targeting ability of nanoprobes. Followed, the target ability of nanoprobes on PDAC cells were quantitatively tested by ICP-OES. Figure 2-Gd-COOH or bTiO2-Gd-IGF1 for 4 h. Similarly, the amount of Ti element of the cells in bTiO2-Gd-IGF1 group is significantly higher than bTiO2-Gd-COOH group, which indicates IGF1 peptide can improve the targeting ability of nanoprobes to MIA PaCa-2 cells. Finally, laser confocal microscope was employed for the observation of rhodamine 123 labeled nanoprobes uptake into MIA PaCa-2 cells. Figure 2-Gd-COOH group, bTiO2-Gd-IGF1 group exhibits more aggregation in the cells, which also indicates IGF1 peptide can improve targeting capability of the nanoprobes.As described above, there are dense fibrous in pancreatic cancer. IGF1R is ubiquitously found in 40–90% of pancreatic cancer cells and stromal cells, which is ideal target sites for nanoprobes to break through the matrix and enter PDAC cells . Here, i2 nanoprobes are less toxic in vitro and in vivo [2 with Gd, dopamine, and IGF1 peptides, so it is still critical to assess nanoprobe toxicity. Figure 2-Gd-COOH and bTiO2-Gd-IGF1 on MIA PaCa-2 cells. Following 24 h’s incubation, the cell viability is higher than 80% in the nanoprobes’ concentration range of 50–300 μg mL−1, which confirms the nanoprobes are low cytotoxicity. The cell survival rate of bTiO2-Gd-IGF1 incubated cells seem to increase when Ti concentration is over 200 μg mL−1. This may be attributed to more nanoprobes entered the cells and the cell color deepened, which affected the absorptions.Previous studies have confirmed bTiO in vivo , 30. Her2-Gd-COOH, bTiO2-Gd-GEM, and bTiO2-Gd-IGF1 or bTiO2-Gd-IGF1-GEM for 2 h. When there is no laser irradiation, cell viabilities are 77.2 ± 0.6% (GEM), 75.5 ± 1.9% (bTiO2-Gd-COOH), 67.0 ± 0.8% (bTiO2-Gd-GEM), 79.5 ± 3.1% (bTiO2-Gd-IGF1) and 68.6 ± 0.3% (bTiO2-Gd-IGF1-GEM), respectively. The results show that only 22.8% of the cells can be killed by GEM after 2 h of incubation, which indicates drug-resistance of the cells. Cell survival rate in bTiO2-Gd-GEM and bTiO2-Gd-IGF1-GEM group is significantly lower than that in bTiO2-Gd-COOH and bTiO2-Gd-IGF1 group, which may be due to the killing effect of GEM. After NIR laser irradiation for 4 min, cell viabilities are 79.4 ± 1.5% (GEM), 75.4 ± 0.5% (bTiO2-Gd-COOH), 75.4 ± 1.6% (bTiO2-Gd-GEM), 46.8 ± 8.6% (bTiO2-Gd-IGF1) and 31.0 ± 8.6% (bTiO2-Gd-IGF1-GEM), respectively. Relative to PTT or chemotherapy alone, killing efficiency in photothermal-chemotherapy group is more significant. In particular, cell killing rate in bTiO2-Gd-IGF1-GEM group can reach 69%.Figure −2 of 808 nm NIR irradiation alone, indicating 808 nm NIR irradiation at 1.2 W cm−2 is safety to cells. However, MIA PaCa-2 cells are killed in varying degrees when the cells were maintained in the same concentrations of GEM, bTiO2-Gd-COOH, bTiO2-Gd-GEM, bTiO2-Gd-IGF1, bTiO2-Gd-IGF1-GEM, and irradiated by 1.2 W cm−2 of 808 nm NIR, while bTiO2-Gd-IGF1-GEM nanoprobes induce more extensive cell death. The live/dead cell staining results are consistent with the MTT results, further indicating that the photothermal-chemotherapy has a more significant killing effect, and can overcome drug-resistance of MIA PaCa-2 PDAC cells.The in vitro photothermal-chemotherapy performance is also visually examined using live/dead cell staining via calcein AM/PI reagents. According to Additional file 2-Gd-COOH, bTiO2-Gd-IGF1 into balb/c mice through tail vein. Additional file 2-Gd-COOH and bTiO2-Gd-IGF1 administered groups. The above results indicate that the prepared nanoprobes offer no toxicity to mice at the doses used for 1 month.The in vivo toxicity assessment of the nanoprobe is carried out by injecting PBS, bTiO2-Gd-IGF1 nanoprobes were injected into the tumor site, and then underwent irradiation with or without 808 nm NIR laser. It can be seen from Fig. 2-Gd-IGF1 on the proliferation of PDAC tumor cells in vivo. Studies have found that IGF1 polypeptide can promote tumor cell proliferation, while Zhou et al. demonstrated IGF1 modified iron oxide nanoparticles could reduce IGF1’s growth-stimulating function, but retain the targeting ability [2-Gd-IGF1 twice, and then analyzed with Ki67 immunohistochemistry. As depicted in Fig. 2-Gd-IGF1 does not enhance cell proliferation index. Therefore, modified bTiO2-Gd-COOH with IGF1 could decrease IGF1’s growth-stimulating activity but still retain targeting ability. Nevertheless, its underlying mechanism is still undetermined.After the mouse pancreatic cancer tumor model was constructed, HE staining was performed on tumor tissue. In Fig.  ability . To furt2-Gd-IGF1-GEM group under NIR irradiation. As depicted in Fig. 2-Gd-IGF1-GEM group. In Fig. 2-Gd-IGF1-GEM group rises from 33.4 to 50.3 °C after NIR irradiation, cancer cells can be rapidly eliminated after only a few minutes of exposure. Hence, the bTiO2-Gd-IGF1-GEM group after NIR irradiation can easily kill cancer cells according to previous study suggested expose of cells in 50 °C [1-weighted MR imaging of PBS is poor, while bTiO2-Gd-IGF1-GEM nanoprobe is better than that of Magnevist at the same Gd concentration, which confirms the feasibility MRI performance of bTiO2-Gd-IGF1-GEM nanoprobes.Followed, photothermal images of tumors are analyzed in PBS group and bTiOin 50 °C . Figure 2-Gd-IGF1 + NIR group was bTiO2-Gd-IGF1 administered, followed by irradiation with NIR. Likewise, the bTiO2-Gd-IGF1-GEM or bTiO2-Gd-IGF1-GEM + NIR group was bTiO2-Gd-IGF1-GEM administered, followed by irradiation without or with NIR, respectively. In order to prove that cooperative photothermal-chemotherapy can loosen matrix barrier and overcome drug-resistance, part of mice were euthanized shortly following the irradiation of NIR, and tumors were assessed via H&E staining. According to Fig. 2-Gd-IGF1 and bTiO2-Gd-IGF1-GEM groups, the green arrows in the figure represent necrosis of tumor tissue. In the bTiO2-Gd-IGF1-GEM + NIR group, most of the cancer cells are eliminate, indicating that advantages of synergetic therapy. Figure 3 in the PBS + NIR, GEM, bTiO2-Gd-IGF1, bTiO2-Gd-IGF1-GEM, bTiO2-Gd-IGF1 + NIR and bTiO2-Gd-IGF1-GEM + NIR groups, respectively. Relative to the bTiO2-Gd-IGF1 + NIR or bTiO2-Gd-IGF1-GEM group, the tumors in bTiO2-Gd-IGF1-GEM + NIR group significantly reduce, which indicate synergetic photothermal-chemotherapy not only can loosen matrix barrier, but also can overcome drug-resistance. The toxic effects of the treatments were evaluated by measuring body weight of the mice within 12 days. In Fig. To evaluate in vivo synergetic photothermal-chemotherapy, the tumor-injected animals were arbitrarily separated into six classes. The PBS + NIR group was PBS administered, followed by irradiation with NIR laser. The GEM group was administered with GEM. The bTiO2-Gd-GEM-IGF1 nanoprobes. First, WB and pathology results confirm IGF1R is ubiquitously found in MIA PaCa-2 cells and their tumor model, meanwhile MIA PaCa-2 cells are resistant to gemcitabine. The IGF1 modified nanoprobes exhibit stronger targeting ability to MIA PaCa-2 cells. Next, cell experiments show that the killing effect of synergistic treatment on MIA PaCa-2-resistant cells is about 3.3 times than chemotherapy alone, which suggest reversal of drug-resistance. Followed, histological experiments prove photothermal can loosen the fibrous matrix of pancreatic cancer model and increase the permeability of blood vessels. Therefore, in vivo results show that tumor models are almost completely cured after 12 days of combined treatments. In conclusion, we provide a more effective approach for treating pancreatic cancer through matrix barrier loosening and drug resistance reverse, and also provide an experimental basis for applying black TiO2 in synergistic photothermal-chemotherapy in pancreatic cancer.In summary, to break matrix barrier and reverse drug resistance in PDAC, this study proposes a dual-targeted photothermal-chemotherapy strategy in PDAC cells and stromal cells, and prepare bTiOAdditional file 1: Figure S1. PH and NIR co-responded drug-release of bTiO2-Gd-IGF1-GEM. Figure S2. The IGF1R expression in MIA PaCa-2 cells, hTERT-HPNE cells, BxPC-3 cells, and Panc-2 cells. Figure S3. Live/dead cell staining following various treatments. Green and red fluorescence denote live and dead cells, respectively. Scale bar is 20 μm. Figure S4. In vivo toxicity analysis of nanoprobes on balb/c mice. The changes of (a) body weight, (b) routine blood indexes, and (c) organ histological analysis after injected with PBS, bTiO2-Gd-COOH, bTiO2-Gd-IGF1 for 1 month. Scale bar is 100 μm.


Scientific Reportshttps://doi.org/10.1038/s41598-022-17113-2, published online 03 August 2022Correction to: The original version of this Article contained errors in Table 2, where the corresponding footnote ‘b’ was omitted in the heading row for ‘β-sandwich (1–139)’, β- ‘barrel 1 (472–583)’ and ‘β-barrel 2 (591–687)’. The correct and incorrect values appear below.Incorrect:Correct:The original Article has been corrected.


H-1,2,4-triazol-3-ato)-6-(1H-πyrazol-1-yl)pyridine} iron(II) complex dimethanol solvate, is a low-spin complex with a moderately distorted pseudooctahedral coordination environment of the metal ion. As a result of the cone shape, the molecules are stacked in mono-periodic columns that are bound by weak hydrogen bonds into di-periodic layers, which, in turn, are arranged in a three-dimensional lattice bound by weak interlayer interactions.The title compound, a charge-neutral bis{2--4 Aea2 structure, the asymmetric unit of the title compound, [FeII(C18H15N6)2]·2MeOH, consists of half of a charge-neutral complex molecule and a discrete methanol molecule. The planar anionic tridentate ligand 2--6-(1H-pyrazol-1-yl)pyridine coordinates the FeII ion meridionally through the N atoms of the pyrazole, pyridine and triazole groups, forming a pseudo-octahedral coordination sphere of the central ion. The average Fe—N bond distance is 1.955 Å, indicating a low-spin state of the FeII ion. Neighbouring cone-shaped molecules, nested into each other, are linked through double weak C—H(pz)⋯π(ph’) interactions into mono-periodic columns, which are further linked through weak C—H⋯N′/C′ interactions into di-periodic layers. No interactions shorter than the sum of the van der Waals radii of the neighbouring layers are observed. Energy framework analysis at the B3LYP/6–31 G theory level, performed to quantify the intermolecular interaction energies, reproduces the weak interlayer interactions in contrast to the strong interaction within the layers. Intermolecular contacts were quantified using Hirshfeld surface analysis and two-dimensional fingerprint plots, showing the relative contributions of the contacts to the crystal packing to be H⋯H 48.5%, H⋯C/C⋯H 28.9%, H⋯N/N⋯H 16.2% and C⋯C 2.4%.As a result of the high symmetry of the  The FeIΣ = Σ112(|90 − φi|), with φi being the N—Fe—N′ angle , with θi being the angle generated by superposition of two opposite faces of the octahedron  is 3.392 Å, angle between planes of the rings is 73.77°]. The formed mono-periodic supramolecular columns protrude along the c-axis with a stacking periodicity equal to 10.6511 (7) Å (= cell parameter c) . Weak intermolecular hydrogen-bonding interactions C—H⋯N/C/O(MeOH) in the range 2.257–2.893 Å . The layers stack along the b-axis direction without any strong or weak interlayer interactions shorter than the sum of the van der Waals radii . The voids between the layers are occupied by methanol molecules, which participate in the strong hydrogen bonding mentioned above, and weak hydrogen bonding with the aromatic substituents within the layers ⋯π(ph’) contacts between the pyrazole (pz) and phenyl (ph) groups, respectively (BF4)2 prepared by dissolving L = 2--6-(1H-pyrazol-1-yl)pyridine  and Fe(BF4)2·6H2O  in boiling acetone, to which chloroform (5 ml) was then added. The middle layer was a methanol–chloroform mixture , which was covered by a layer of methanol (10 ml), to which 100 µl of NEt3 was added dropwise. The tube was sealed, and black plate-like single crystals appeared within 3-4 weeks (yield ca 75%). Elemental analysis calculated for C38H38FeN12O2: C, 60.80; H, 5.10; N, 22.39. Found: C, 60.50; H, 5.31; N, 22.71.The ligand 8.Uiso(H) = 1.2–1.5Ueq(C); the hydrogen atom H26 was refined freely. Two OMIT commands were used to exclude beamstop-affected data.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022009744/yz2021sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022009744/yz2021Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022009744/yz2021Isup5.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022009744/yz2021sup4.docSupporting data for energy framework analysis and schematic structures of related complexes. DOI: 2211089CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


K. marxianus is a promising cell factory for producing heterologous proteins. Oxidative stresses were raised during overexpression of heterologous proteins, leading to the shift of the redox state. How to measure the redox state of live K. marxianus cells without perturbing their growth remains a big challenge. Here, a fluorescence lifetime imaging (FLIM)-based method was developed in live K. marxianus cells. During the early exponential growth, K. marxianus cells exhibited an increased mean fluorescence lifetime (τ-mean) of NAD(P)H compared with Saccharomyces cerevisiae cells, which was consistent with the preference for respiration in K. marxianus cells and that for fermentation in S. cerevisiae cells. Upon oxidative stresses induced by high temperature or H2O2, K. marxianus cells exhibited an increased τ-mean in company with decreased intracellular NAD(P)H/NAD(P)+, suggesting a correlation between an increased τ-mean and a more oxidized redox state. The relationship between τ-mean and the expression level of a heterologous protein was investigated. There was no difference between the τ-means of K. marxianus strains which were not producing a heterologous protein. The τ-mean of a strain yielding a high level of a heterologous protein was higher than that of a low-yielding strain. The results suggested the potential application of FLIM in the non-invasive screen of high-yielding cells. K. marxianus is a promising microbial cell factory for producing heterologous proteins, bioethanol and bulk chemicals H/NAD(P)+ . Optical+ . Using N+ . Fluores+ . The rat+ . a1/a2 iof cells . Usuallyof cells . Howeverus cells .K. marxianus cells was assessed by FLIM of NAD(P)H. K. marxianus cells exhibited an increased τ-mean of NAD(P)H compared with S. cerevisiae cells during early exponential growth, which was consistent with different Crabtree-effect statuses of two yeasts. K. marxianus cells exhibited an increased τ-mean upon oxidative stresses induced by high temperature or H2O2, suggesting a correlation between an increased τ-mean and a more oxidized redox state. Notably, an increased τ-mean was correlated the high-level expression of a heterologous protein in K. marxianus cells, which suggested a potential application of FLIM in the non-invasive screen of high-yielding cells.In this study, the redox state of K. marxianus strain FIM-1  at 30°C overnight and the culture was diluted into a fresh YPD medium to start at an OD600 of 0.1. Cells were then grown at 30°C for 3 h. To compare redox states at different temperatures, overnight culture of FIM-1 was inoculated into a fresh YPD medium to start at an OD600 of 0.1 and then grown at 30°C or 45°C for 24 h. To perform the H2O2 treatment, FIM-1 cells were grown at 30°C for 24 h and then grown in the presence of 20 mM H2O2 for 1 h. Untreated culture served as a control. To compare redox states during expressing IBDV-VP2, KML and KMH cells were grown in the YD medium  for 3 days. Cells were collected after 1 or 3 days.To compare the redox states of The culture of FIM-1 and S288C was centrifuged and the supernatant was subjected to analysis. The amount of dissolved oxygen was measured by a dissolved oxygen analyzer  according to the manufacturer’s manual. Residual glucose and ethanol was measured on high-performance liquid chromatography  as described before .600 of 5. A total of 5 μl sample was transferred to the ConA-coated dish for analysis.To immobilize yeast cells, 20–25 μl of ConA  was spread onto the bottom of a glass-bottom petri dish  and the dish was left to dry overnight at room temperature. Yeast cells were pelleted and resuspended in sterile water to an ODt-test. Otherwise, Welch’s modified unpaired t-test was used.FLIM was performed by a Leica TCS SP8 DIVE FALCON microscope  equipped with a ×63 water immersion objective (N.A. = 1.2) excited by a femtosecond pulsed laser with ∼3 mW power at 740 nm . The FLIM images were exported in lif format and ROIs were selected for processing in Leica Application Suite X software to obtain fluorescence lifetime data. The sizes of experimental ROIs samples ranged from 52 to 104. The data were fitted using a double exponential model as τ-mean = a1 τ-free + a2 τ-bound . Statist+, NADH, NADP+ and NADPH were determined by a WST-8 based colorimetric assay using commercial kits . To collect cells grown at different temperatures and cells treated with H2O2, FIM-1 cells were cultured as described above in “Medium and culturing”. Cells of 20 OD600 were collected, washed and then suspended in 200 μl extraction buffer provided by the kit. Cells were then mixed with 200 μl acid-washed glass beads  and processed by a bead-beater  at 6 m/s for 2 min. The lysate was centrifuged at 13,200 rpm for 10 min at 4°C. A total of 20 and 50 μl supernatant was collected for NAD+/NADH and NADP+/NADPH assay, respectively. Experiments were performed in triplicates and repeated three times. Ratios of NADH/NAD+, NADPH/NADP+ and NAD(P)H/NAD(P)+ were calculated based on the concentrations.Concentrations of intracellular NAD+  and K. marxianus cells (FIM-1) during the early exponential growth were investigated in the first place. After 3 h of growth in the YPD medium (containing 20 g/L glucose), the OD600 of K. marxianus cells was 1.49 times that of S. cerevisiae cells H in K. marxianus and S. cerevisiae cells were quantified as 1.21 ± 0.27 ns and 0.94 ± 0.19 ns, respectively (K. marxianus cells (2.23) was significantly lower than that in S. cerevisiae cells (3.40) (K. marxianus cells was relatively more oxidized. The lower a1/a2 of K. marxianus cells was mainly due to significantly increased τ-bound of NAD(P)H H, as a longer τ-mean of K. marxianus cells suggested a more oxidized state.To compare the redox states of two yeasts, the fluorescence lifetime of NAD(P)H in cells was analyzed by FLIM. In the pseudocolor mapping of NAD(P)H lifetime, ght blue , suggestectively , left. Ts (3.40) , middle, NAD(P)H , right, 2O2 induced oxidative stress in K. marxianus  were grown at 30°C or 45°C for 24 h. Pseudocolor mapping of NAD(P)H lifetime of cells at 30°C showed dark blue and faint cyan, indicating a shorter lifetime  was significantly longer than that in untreated cells (1.04 ± 0.07 ns) H of cells treated with 20 mM H0.07 ns) , left. τ0.07 ns) , right. 0.07 ns) , middle,+, NADPH and NADP+ were determined by a colorimetric chemical assay. As shown in +, NADPH/NADP+ and NAD(P)H/NAD(P)+ of cells grown at 45°C were all significantly decreased, compared with their counterparts of cells grown at 30°C. Similarly, H2O2 treatment significantly decreased NADH/NAD+, NADPH/NADP+ and NAD(P)H/NAD(P)+ of cells (2O2 (K. marxianus cells could be applied to indicate a shift in redox state [defined by NAD(P)H/NAD(P)+] toward a more oxidative status.To confirm the relationship between NAD(P)H content and τ-mean, concentrations of intracellular NADH, NADof cells , right. lls  capsid VP2 by an episomal vector   , left. Cid cells , middle.void (1.02 ± 0.09 ns) and KMLvoid cells (1.03 ± 0.08 ns). The distribution of τ-mean of KMHvoid and KMLvoid cells was more concentrated. It suggested that, after entering the stationary phase, cells without expressing a heterologous protein exhibited a redox state with less fluctuation. Consistent with the highest τ-mean of KMH cells, a1/a2 of KMH cells was the lowest and τ-bound of KMH cells was the longest among their counterparts H (Since Britton Chance discovered the autofluorescence of NAD(P)H in the 1950s , methods NAD(P)H . Therefo NAD(P)H . The red NAD(P)H . Yeast i NAD(P)H . FLIM isK. marxianus cells upon various oxidative stress. Among the current methods to analyze the redox states of cells, chemical analysis destroys cell integrity and does not allow for single-cell analysis (2O2 treatment led to an increase of τ-mean of NAD(P)H. But H2O2 caused more deviation of τ-mean than high temperature, suggesting more fluctuated redox states were raised among the cells treated by H2O2 H H . Therefo NAD(P)H . For exa NAD(P)H . However NAD(P)H . Since F NAD(P)H . If a mo


Scientific Reportshttps://doi.org/10.1038/s41598-019-56612-7, published online 30 December 2019Correction to: The original version of this Article contained an error in Figure 5 (a) label, where the stereochemistry label was incorrect.“-8b”now reads:“-8b”The original Figure The original Article has been corrected.


Candida infection. A simple and effective DNA preparation method is of critical for standardizing and applying molecular diagnostics in clinic laboratories. The goal of this study was to develop a Candida DNA preparation method that was quick to do, easy to perform, and bio-safe. Snailase and lyticase were screened and combined in this work to enhance the lysis of Candida cells. The lysis solution composition and metal bath were optimized to boost amplification efficiency and biosafety. A duplex real-time PCR was established to evaluate the sensitivity and specificity of the preparation method. Using the supernatant from the rapid preparation method as templates, the duplex PCR sensitivities for five common Candida species were determined to be as low as 100 CFUs. When compared to conventional preparation methods, the samples prepared by our method showed higher PCR detection sensitivity. PCR identification and ITS sequencing were 100% consistent, which was better than biochemical identification. This study demonstrates a rapid method for Candida DNA preparation that has the potential to be used in clinical laboratories. Meanwhile, the practical application of the method for clinical samples needs to be proven in future investigations.Nucleic acid based molecular technologies are the most promising tools for the early diagnosis of The online version contains supplementary material available at 10.1186/s13568-022-01500-z. Candida species are among the top four pathogens responsible for nosocomial bloodstream infection , including candidemia, is a significant cause of morbidity and death, particularly in individuals with severe medical conditions , hybridization, and the loop-mediated isothermal amplification (LAMP), have been shown to identify the infection even in its early stages  medium  and harvested by centrifugation at 400 × g for 5 min. After two washes with PBS, the fungal density was determined using a cell counting plate. A Candida count of 5 × 107 CFUs was used to choose the suitable digestive enzymes; a Candida count of 5 × 105 CFUs was utilized to validate the ideal lysis conditions, which may be closer to the fungal load in real applications.Candida cells. Aliquots of enzyme at different concentrations were prepared according to the manufacturer’s instructions.Digestive enzymes, including snailase , lyitcase , zymolyase  and glucanase , were tested for their capacity to lyse Candida cells were resuspended in 1 mL of suspension buffer  containing 2 μL of β-mercaptoethanol. The different digestive enzymes then were added to each Eppendorf tube at the following concentrations: (1) snailase: 0, 130, 650, 1040 and 2080 μg/mL; (2) lyitcase: 0, 100, 200, 500 and 800 U/mL; (3) zymolyase: 0, 40, 80, 160 and 320 U/mL; and (4) glucanase: 0, 40, 80, 160 and 320 U/mL. All of the digestive reaction systems described above were incubated at the temperatures recommended by the enzymes, which were 35, 30, 30 and 50 °C for snailase, lyitcase, zymolyase and glucanase, respectively. The lysing effects were assessed after 5 h by counting the remaining intact Candida blastospores with a cell counting plate.The precipitated C. albicans SC5314 cells were collected by centrifugation. Following a PBS wash, the cells were treated with a yeast lytic enzyme kit . SC5314 cells were resuspended in 470 µL of sorbitol buffer  and incubated for 10 min at room temperature. Then, 25 µL of enzyme solution and 5 µL of β-mercaptoethanol were added into the SC5314 suspension. The mixture was incubated in a water bath at 30 °C for 4 h before being centrifuged at 14,000 g for 30 min. The genomic DNA was extracted from the collected precipitates using the Yeast DNA Extraction Kit  according to manufacturer’s instructions.Overnight cultured WE designed an interference experiment to evaluate the impact of the components on the PCR because the lysis solution comprises a variety of components. The purified SC5314 genomic DNA was used as the PCR template. Snailase, lyticase, β-mercaptoethanol, sorbitol and EDTA were separately added into the 50 µL PCR reaction systems to observe their effects on amplification. Primer and probe sequences were as follows: forward primer, 5′- GGG TTT GCT TGA AAG ACG GTA -3′; reverse primer, 5′- TTG AAG ATA TAC GTG GTG GAC GTT A -3′; TaqMan probe, 5′-FAM- ACC TAA GCC ATT GTC AAA GCG ATC CCG -TAMRA-3′. PCR reactions were performed in 50 µl volumes, containing 2 µl of each primer (forward and reverse) at a concentration of 10 µmol/L, 25 µl of 2 × Taq PCR Mix , 2 µl Taqman probe, 4 µl of DNA template (∼50 ng) and a specified volume of lysate components; The reaction volume was adjusted to 50 μL with double-distilled water. The qPCR was performed using a SLAN 96P real time PCR System , and cycling conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min.C. albicans SC5314 cells were washed with PBS and divided into two equal aliquots. Following centrifugation, the aliquots were incubated with optimized lysis solution at 30 °C for 1 h. Then, one aliquot was heated at 100 °C in a DB100C metal bath  for 10 min, while the second aliquot was left untreated. After centrifuging the lysates for 5 min, the supernatants were utilized. The parameters of the amplification curve and cycle threshold (Ct) values were used to assess the effect of high temperature on PCR performance.Cultured Candida species. The primers and probes were designed as previously reported  values and lower Ct values (19.08 ± 0.06 VS 20.26 ± 0.11) when compared to untreated samples.As shown in Fig. Candida spp.’s cultured blastospores were collected and adjusted to indicate densities. The samples were treated with a high temperature metal bath after being incubated with the optimized lysis solution. When lysate supernatants were used as templates for PCR amplification, the sets of specific primers and probes were able to effectively amplify the corresponding Candida samples, with detection limits as low as 100 CFUs  were varied when detecting different Candida species, which was 101 CFUs for C. glabrata and C. parapsilosis and 100 CFUs for C. albicans, C. tropicalis, and C. krusei  the incidence rate of C. albicans and C. tropicalis infection is relatively high, and the detection system for them is mature . (A)negative control, (B)130µg, (C)650µg, (D)1040µg, (E)2080µg. Figure S2. Digestive effect of snailase on Candida albicans (400×). (A)negative control, (B)100U, (C)200U, (D)500U, (E)800U. Figure S3. Digestive effect of zymolyase on Candida albicans (400×). (A)negative control, (B) 5U, (C) 20U, (D) 50U, (E) 100U. Figure S4. Digestive effect of glucanase on Candida albicans (400×). (A)negative control, (B) 40U, (C) 80U, (D) 160U, (E) 320U. Figure S5. Combination use of snailase & lyticase (400×). (A)negative control, (B) 13µg snailase+2U Lyticase, (C) 65µg snailase+10U Lyticase, (D) 130µg snailase+20U Lyticase, (E) 650µg snailase+100U Lyticase, (F) 1040µg snailase+200U Lyticase. Figure S6. PCR interference experiment that adding different lysis solution components into the 50 µl PCR reaction system. (1) positive control, (2) 16 µg snailase, 2.5U Lyticase, (3) 0.05 µl β-mercaptoethanol, (4) 12.5mM sorbitol, (5) 0.625mM EDTA, (6) negative control. Figure S7. Optimized lysis solution composed of snailase, lyticase and β-mercaptoethanol could totally disrupt of blastospores C. albicans (A), C. tropicalis (B) and C. parapsilosis (C). However, has no effect on blastospores of C. glabrata (D) and C. krusei (E). Figure S8. Sensitivity of multiple PCR (specific primers and probes). (A) Candida albicans, (B) Candida tropicalis, (C) Candida parapsilosis, (D) Candida glabrata, and (E) Candida krusei. The numbers of 0-5 represent Candida density of 100-105CFU/ml, and NC represents a negative control. Figure S9. Sensitivity of multiple PCR . (A) Candida albicans, (B) Candida tropicalis, (C) Candida parapsilosis, (D) Candida glabrata, and (E) Candida krusei. The numbers of 0-5 represent Candida density of 100-105CFU/ml, and NC represents a negative control. Figure S10. Specificity of duplex PCR. The amplification curve of A-E were: (A) Candida. albicans, (B) Candida. tropicalis, (C) Candida. parapsilosis, (D) Candida. glabrata, (E) Candida. krusei. (F), The curves by Candida universal primer to amplify five common Candida species: (1) Candida. tropicalis, (2) Candida. parapsilosis, (3) Candida. krusei, (4) Candida. albicans, (5) Candida. glabrata.


A face-to-face stacking of aromatic rings is an effective non-covalent strategy to build functional architectures, as elegantly exemplified with protein folding and polynucleotide assembly. However, weak, non-directional, and context-sensitive van der Waals forces pose a significant challenge if one wishes to construct well-organized π-stacks outside the confines of the biological matrix. To meet this design challenge, we have devised a rigid polycyclic template to create a non-collapsible void between two parallel oriented π-faces. In solution, these shape-persistent aromatic clips self-dimerize to form quadruple π-stacks, the thermodynamic stability of which is enhanced by self-complementary N–H···N hydrogen bonds, and finely regulated by the regioisomerism of the π-canopy unit. With assistance from sufficient electrostatic polarization of the π-surface and bifurcated hydrogen bonds, a small polyheterocyclic guest can effectively compete against the self-dimerization of the host to afford a triple π-stack inclusion complex. A combination of solution spectroscopic, X-ray crystallographic, and computational studies aided a detailed understanding of this cooperative vs competitive process to afford layered aromatics with extraordinary structural regularity and fidelity. Stacking aromatic molecules through π–π interactions is a useful strategy to create ordered layers of small molecules, but the weak and non-directional nature of these forces creates a challenge in achieving long-range structural ordering. Here, a rigid molecular template that facilitates the creation of a non-collapsible void between two parallel π-faces is designed, and a central N–H motif is shown to increase the thermodynamic stability of self-dimerized assemblies and host–guest complexes via N–H…N hydrogen bonds. To define efficient pathways for charge or energy carriers, precise molecular-level control of the π–π stacking is needed15. For example, a linear molecular backbone can support regularly spaced aromatic branches that define a tight π-stack, as exemplified with comb-like polymers19, hairpin turn-motifs25, and multi-layer cyclophanes27. Despite intuitive appeal in design, with flexible polymers, it is not easy to control the relative orientations of the planar π-branches along the backbone axis17. For small molecules, the spacing between individual π-faces might be controlled precisely, but covalent construction of multi-layer molecules to achieve long-range structural ordering is a synthetically demanding task.Electronically tunable columnar stacks of aromatic molecules33. Prominent examples include aromatic guest-encapsulated molecular cages38, through-space π-conjugated foldamers41, metal coordination-induced π-stacking44, and self-assembled molecular tweezers49. To control the special ordering and orientation of π-faces with high fidelity, a rational molecular design strategy is needed54. In this paper, we report the chemistry of shape-persistent molecular clips.To overcome these challenges, supramolecular systems have been developed by using non-covalent interactions between small-molecule building blocks56, only a a–b–b–a type π-sequence, not an isomeric a–a–b–b or b–a–a–b, is allowed for the quadruple layer57. The regularity in stacking orientation59 and stacking sequence59 facilitate the self-association of quadruple π-stacks into an infinite columnar π-stacks in the solid state. We also demonstrate that the quadruple π-stacks can disassemble into individual π-clips to encapsulate a small polyheterocyclic molecule, thereby affording a guest-intercalated a–c–b type triple π-stacks. Such structure- and context-dependent Tetris-like assembly is a testament to the importance of individually weak yet collectively strong non-covalent interactions, the details of which are provided in the following sections.As schematically shown in Fig. a and b in a parallel fashion, while leaving a non-collapsible void in between. In addition to functioning as a rigid structural support, the indole unit provides an N–H group as a hydrogen-bonding donor (HBD). The intermolecular N–H···N hydrogen bonds ensure that only a a–b–b–a type π-sequence takes full advantage of the shape complementarity and the maximum number of hydrogen bonds.To build rigid clip-like molecules that self-assemble into anti-parallel π-stacks Fig. , a spiroC-NI, C-P1, C-P2, and C-P4 were designed. While sharing the common C-shaped skeleton, these molecules differ in the chemical composition  and regiochemistry  of the upper canopy unit .Using this general structural platform, a homologous set of molecular clips 1 with different regioisomers of pyrene boronic ester/acids to prepare 2  was subjected to a condensation reaction with the common precursor 5 to afford the corresponding aminals 6. Unlike ketals, aminals are prone to hydrolysis back to ketone and amine61; few isobenzimidazole are thus known to have aromatic substituents at the quaternary carbon center63. For 6, two intramolecular N–H···N hydrogen bonds .The synthesis of the upper part of the π-clip required direct C–C cross-coupling reactions of cyclic ketone-fused bromoindole 1H NMR spectroscopic studies of C-NI, its N-methylated derivative C-NIMe, and the substructure model 7  of the naphthyl, pyridyl, and isobenzimidazolyl C–H protons  Å) buried deep inside the hydrophobic cavity  and isobenzimidazole···isobenzimidazole (= b···b) contacts, respectively , and the mean centroids of the two stacked (and thus facing) pyridine–isobenzimidazole–pyridine triads (for b···b).A subsequent single-crystal X-ray diffraction (SC-XRD) study on ]2 Figs. . Upon seity Fig. . When viity Fig. , the quab–a Fig. . The intely Fig. , right, C-NI]2 stack further to afford an infinite one-dimensional column with an inter-dimer π–π stacking distance of 3.187(2) Å  were determined as 8.1 (±2.3) × 102 M−1 for C-P1, and 1.9 (±0.3) × 102 M−1 for C-P2 in CDCl3  × 102 M−1 was obtained for C-P2  revealed that the solvent-accessible surface areas are essentially identical for monomeric C-P1 and C-P2  × 102 M−1), the 1H NMR spectrum of a dilute (2.4 × 10−2 mM) sample of C-P2 is similar to that of the non-stacking model 7 and the N-methylated derivative C-P2Me  to fit the isotherm  × 105 M−1 is sufficiently large so that the solution population is dominated by the dimeric [C-NI]2. The variable temperature (VT)  and overnight scanning barely shifted the pyridine resonance  × 105 M−1 is the lower limit of the self-dimerization affinity of C-NI. Similar to the situation of C-P2 showing an enhanced dimerization propensity in more polar CD2Cl2  × 105 M−1 in CD2Cl2  Fig.  NMR specVT) Fig.  data corC-NI was further manifested in comparative studies with C-P2. In DMSO-d6 at T = 25 °C, the 1H NMR spectrum of C-P2 revealed the co-existence of monomer and dimer  map revealed the electron-deficient nature of naphthaleneimide  in CDCl3 provided diffusion coefficients of 8.80–9.55 × 10−10 m2 s−1, which are smaller than that of monomeric C-NIMe (1.13 × 10−9 m2 s−1), but indicate no significant inter-dimer interactions in the solution state  Å and 3.8182(17) Å, and dissymmetric bifurcated hydrogen bonds with dN–H···N = 2.947(5) Å and 3.442(5) Å  have a flat and large π-surface to fit inside the interplanar void, and (ii) form hydrogen bond(s) with the indole N–H group inside the cavity. Among various small molecules  Å Fig. .Fig. 7Ho3 at r.t., a mixture of C-P2 and PHD produced large chemical shifts in the aromatic proton resonances of both C-P2 and PHD  × 102 M−1  × 102 M−1 was estimated for C-P1⊃PHD , phenanthrene, 1,10-phenanthroline (phen), and 2,2′-bipyridine (bpy). As shown in Fig. C-P2, either  × 103 M−1  relative to C-P2 (µ = 1.54 D), thus engendering stronger antiparallel dipole–dipole interaction with entrapped PHD (µ = 2.61 D). A competitive encapsulation of the aromatic guest thus turns a–b–b–a type quadruple π-stacks into a–c–b type triple π-stacks  and only a small amount of free C-NI (4%) (vide supra). The addition of the PHD guest (6 equiv) to this system reshuffles the π-stacks, from which the triple-decker C-NI⊃PHD would emerge as the dominant species (97%), equilibrating with only tiny fractions of the quadruple-decker [C-NI]2 (2%) and unbound C-NI (1%). This molecular redistribution process can be graphically represented by the speciation plot in Fig. Under similar conditions, the addition of PHD to 2 and [C-NI]2, crystallographically determined atomic coordinates were used as an input without a geometry optimization process. The calculated structures were varied by frequency calculations: no imaginary frequencies for the minima. Molecular electrostatic potential (MEP) maps were generated by using GaussView 5 and Gaussian 09 software.All DFTPeer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie


Thus, extensive investigations into efficient energy storage systems for promoting the utilization of clean and renewable energy have been greatly stimulated as the research focus worldwide. Lithium ion batteries (LIBs), as the most promising energy storage devices, have more and more requirements  for advanced electrode materials.4 Typically, metal-catecholates (M-CATs), a class of conductive MOFs, are composed of central metal ions and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) ligands,5 where metal ions are coordinated with adjacent HHTP linkers to form an extended two-dimensional (2D) framework. Meanwhile, the oxygen atoms in HHTP can also combine with axial water ligands to form hydrogen bonds.5 Thus, M–CAT exhibits two types of stacking structures; one is the 2D extended framework with hexagonal pores (∼1.2 nm) and a honeycomb structure via oxygen bonds and π–π interactions, where metal nodes and organic linkers serving as charge carriers enable full charge delocalization in the 2D plane, resulting in good electrical conductivity,6 and the other is the stacking along the c-axis via hydrogen bonds, which forms a connection between the layers, easily forming one-dimensional (1D) structures.5 Owing to its unique porous structure and good electrical conductivity, the M–CAT holds enormous promise in wide energy-related applications, such as catalysis,7 supercapacitors8 and LIBs.9 However, the intrinsic charge-storage mechanisms of the M–CAT electrodes have rarely been studied so far.Recently, conductive metal organic frameworks (MOFs) have drawn enormous attention as potential electrode materials because of their simple synthesis process, adjustable structure, environmental amity, and especially superb electronic conductivity.DLi values in the order of 10−9 to 10−10 cm2 s−1 during the intercalation/de-intercalation process and delivered a large discharge capacity of ∼889 mA h g−1 at 0.1 A g−1 as well as good rate capability with ∼428 mA h g−1 at 2.0 A g−1 and remarkable cycling stability even at high rates.In this work, we reported the hydrothermal synthesis  is combined with two HHTP molecules to form a hexagonal honeycomb structure. The porosity of Ni-CAT NRs is evaluated by Brunner–Emmett–Teller (BET) measurements  analysis reveals the good crystalline character of the Ni-CAT NRs, as depicted in θ = ∼4.7, ∼9.5, ∼12.6 and ∼16.5° illustrate the long-range order of the crystal in the ab plane, and the distinct reflection at ∼27.3°, corresponding to the (001) plane, implies the short-range order of the c axis, indicating that the resultant Ni-CAT is a covalently linked layered material.11The typical crystal structure of Ni-CAT, as shown in −1 are attributed to the –C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C– stretching, –C–O– stretching vibration, and –O–H, respectively,12,13 fully proving the existence of multiple organic functional groups related to organic HHTP. Meanwhile, the X-ray photoelectron spectroscopy (XPS) full-scan spectrum  peaks at 861.4 and 879.7 eV, indicating the presence of bivalent Ni in the Ni-CAT NRs.12 Besides, the O 1s spectrum can be deconvoluted into three peaks at 531.0, 532.4 and 533.3 eV, which represent O–Ni, OC and O–C, respectively.12 The detailed physicochemical characterization and analysis above confirm the successful formation of the 1D Ni-CAT NRs.In order to further analyze the phase structure of Ni-CAT, Fourier transform infrared spectroscopy (FT-IR) was conducted, as presented in 2 The galvanostatic intermittent titration technique (GITT) is an effective and credible approach to evaluate the solid phase diffusion properties.2−1, where the charge time (t) is 200 s and the rest time (τ) is 150 s ,10 N-doped yolk–shell carbon nanocages filled with ZnSe/CoSe2 nanodots (∼10−15 cm2 s−1),14 hollow NiO@Co3O4@graphene quantum dot spheres (∼10−15 cm−2 s−1),15 ZnCo2O4@carbon nanotubes (∼10−11 cm−2 s−1),16 and so on. Notably, the changing trend of the DLi values during charging  at a scanning rate of 0.1 mV s−1  in the c-axis direction,21 as confirmed by the ex situ XRD data  benzene rings, (II) pores and (III) interlaminar space  of ∼68%. The capacity loss here can be ascribed to the formation of the SEI film and the irreversible Li extraction process.19 Additionally, the discharge capacities of the 2nd and 3rd cycles still remain as large as ∼1010 and ∼993 mA h g−1, respectively.−1 at different current densities of 0.1, 0.2, 0.5, 1.0, 1.5 and 2.0 A g−1, respectively. When the current decreases in turn to 0.1 and 0.2 A g−1, the reversible capacities can be rapidly recovered to ∼652 and ∼648 mA h g−1, respectively. As depicted in −1 at 2.0 A g−1),22 Co-isophthalic MOF (∼157 mA h g−1 at 0.5 A g−1),23 Pb2 (∼268 mA h g−1 at 2.0 A g−1),24 Ni/Mn-1,3,5-benzenetricarboxylic acid (∼270 mA h g−1 at 1.5 A g−1),25 Cdtripiperidine-4-carboxylic acid) (∼200 mA h g−1 at 1.0 A g−1),26 Co3(HHTP)2 (∼380 mA h g−1 at 2.0 A g−1),27 and Cu-CAT (∼381 mA h g−1 at 2.0 A g−1).28 This convincingly highlights the remarkable advantages of 1D porous Ni-CAT NRs for high-power LIBs, thanks to their intrinsically superb Li diffusion and electronic conductivity.29,30Besides, the rate performance of the Ni-CAT anode is shown in −1. Appealingly, the Ni-CAT NRs maintain a reversible capacity of ∼626 mA h g−1 after 200 cycles at 0.2 A g−1. More strikingly, the NR electrode can even maintain a capacity as large as ∼592 mA h g−1 after the same number of cycles at a high current rate of 0.5 A g−1. Moreover, the CE values for each cycle approach 100% except for the first cycle. Meanwhile, the structure of Ni-CAT NRs is relatively stable even after 300 cycles. The TEM observations (Fig. S7, ESIab plane remains stable, and that only the c axis is somewhat damaged, which makes the length of Ni-CAT NRs further shortened, in good agreement with the ex situ XRD analysis above (Fig. S6, ESI+-storage mechanism of Ni-CAT was rationally proposed. The electrically conductive Ni-CAT NRs were appealingly endowed with a high Li+ diffusion coefficient. As a result, the as-synthesized Ni-CAT NRs exhibited large reversible capacities of ∼889 mA h g−1 at 0.1 A g−1 and even ∼428 mA h g−1 at a high rate of 2.0 A g−1, and exceptional long-term cycling behaviour with a capacity of ∼592 mA h g−1 maintained over 200 consecutive cycles at 0.5 A g−1. The work will inspire future versatile design and construction of novel conductive MOFs for energy-related applications.In summary, the investigation here mainly reports the hydrothermal synthesis of 1D porous conductive Ni-CAT NRs as anode materials for high-performance LIBs. More significantly, the underlying LiThere are no conflicts to declare.NA-001-C9NA00616H-s001


N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.  •Peptidoglycan consists of N-acetylglucosamine, N-acetylmuramic acid, and amino acids•We developed a one-step synthesis of azide-labeled UDP-N-acetylglucosamine•In vivo generated azide-labeled UDP-N-acetylglucosamine gets incorporated into peptidoglycan•Bacteria were fluorescently labeled on N-acetylglucosamine of peptidoglycan Chemistry; Biosynthesis; Biomolecules; Glycobiology N-Acetylglucosamine (GlcNAc or NAG) is an essential monosaccharide required in almost all organisms. In bacteria, GlcNAc is a key component of several important cell envelope molecules, including lipopolysaccharide (LPS) (N-acetylglucosamine (PNGA) (N-acetylmuramic acid (MurNAc) sugars connected by short peptides attached to the lactoyl acid residue of MurNAc  , peptidode (LPS) , wall tede (LPS) , enterobde (LPS) , and pole (PNGA) . The PG e (PNGA) . The PG f MurNAc . The cytf MurNAc , called f MurNAc . The finf MurNAc   and subsequent analysis indicated that the labels were incorporated into the peptidoglycan layer.Traditional chemical synthesis of  sugar-nucleotides has been reported over past decades , whereas barrier . A varia barrier . Moreove barrier . Herein,1. By using acetyl chloride as solvent and reaction substrate to produce compound 1, the acetylation and chloride-substitution are achieved in one step. The resulting product reacted with 2-chloro-4-nitrophenol in the presence of triethylamine to the 2-chloro-4-nitrophenyl glycoside. The final product, 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP), was synthesized by introduction of the azido group and removal of the acetyl groups.We first set out to optimize the synthesis of the activated sugar (GlcNAz-CNP) such that it would allow the facile synthesis of relatively large amounts. To synthesize an aromatic glycoside, the targeted hydroxyl group of the sugar needs to be activated by introducing a halide . In this4. The glycosyltransferase reaction was followed by measuring the appearance of yellow color due to the production of 2-chloro-4-nitrophenolate (CNP) during the reaction , indicating that this reaction may require different substrate concentrations or other conditions for optimal conversion to Lipid II. However, we could successfully synthesize GlcNAz-labeled Lys- or m-DAP-Lipid II by the reaction of UDP-GlcNAz and the corresponding type of Lipid I in a reaction catalyzed by a Micrococcus flavus membrane preparation (data not shown for Lys-Lipid II) (m/z 2246.24 Da ([M−H]-)  A and S2Bipid II) D, mass s([M−H]-) B and S2C([M−H]-) .E. coli PBP1B (PBP1BEc), which interacts with proteins from the cell division machinery and is a major contributor to the synthesis of septal PG (Ec was unable to polymerize dansyl-GlcNAz-Lipid II (Ec polymerized Lipid II labeled with dansyl at the lysine of the pentapeptide (dansyl-lysine-Lipid II), as expected   B. We alsLipid II C. This iin situ by providing GlcNAz-CNP to E. coli cells expressing OleD. We hypothesized that GlcNAz-CNP is converted to UDP-GlcNAz by OleD, using the endogenous pool of UDP. UDP-GlcNAz can then be used to synthesize Lipid II, which in turn will be used for the synthesis of PG. To test this hypothesis, we expressed OleD in BL21(DE3) cells and added GlcNAz-CNP to the growth medium. In principle, the outer membrane of E.coli is permeable to a range of small molecules and sugars  that was not present in control digestions (Ⅱ). This proves the presence of GlcNAz in the isolated PG. MS/MS analyses of this molecule supported the identity of the GlcNAz-labeled disaccharide tetrapeptide monomer  as the loss of GlcNAz from the parent ion results in an m/z = 737.346 or, with an extra neutral loss of water, an m/z = 719.337. Together, these results confirm that GlcNAz was specifically incorporated into bacterial PG. GlcNAz-CNP therefore can be converted into UDP-GlcNAz by heterologous OleD and the resulting precursor is used in the biosynthesis of PG. In our experiments, we did not observe azido MurNAc-containing compounds even though UDP-GlcNAc is a precursor of UDP-MurNAc in the bacterial cell. Most likely, the specificity of enzymes required for the UDP-MurNAc synthesis may be too high and cannot use UDP-GlcNAz as a substrate. This labeling method could likely be applicable to other bacteria, provided that OleD can be heterologously expressed.Next, we aimed to obtain more direct evidence for the presence of GlcNAz in PG. For this, we isolated PG from cells expressing OleD and grown in the presence of GlcNAz-CNP. We digested the PG with the muramidase cellosyl to produce the disaccharide peptide subunits (muropeptides) and analyzed the muropeptides with nano-LC coupled to a mass spectrometer , 2015. Wgestions C trace ⅡBy expressing the heterologous glycosyltransferase OleD in bacteria, we could incorporate the unnatural amino sugar GlcNAz into the PG layer. Given the promiscuity of OleD for the sugar nucleotide donor , this laOur study is limited to labeling bacteria (or other microorganisms) that express the OleD enzyme or homologs thereof. Lipid II labeled with a fluorophore at the N-acetylglucosamine cannot be used to study the glycosyl-transferase activity of PBPs.E.J.Breukink@uu.nl).All requests for reagents and resources should be directed to the lead contact, Eefjan Breukink  pLysS transformed with pET28a-OleD-TDP16 plasmid was used for the production of OleD and the in vivo incorporation of GlcNAz into the peptidoglycan layer. Experiments were conducted at 37°C unless otherwise noted. All information on alternate temperature conditions, as well as media conditions for experiments, can be found in the 254, SO2). Iodine (Merck) staining also was used for visualization of TLC. UDP-MurNAc-pentapeptide extract was obtained according to the previously reported method  grade or finer and were used without further purification unless otherwise stated. The compounds were purified by flash silica column chromatography . All reactions and fractions from column chromatography were monitored by thin-layer chromatography (TLC) using plates with or without a UV fluorescent indicator , number of protons, and coupling constants (J) in Hertz (Hz). When appropriate, the multiplicity is preceded by br, indicating that the signal was broad. 13C NMR spectra were recorded at 100 or 125 MHz with chemical shifts reported relative to CDCl3 δ 77.0. High-resolution mass spectrometry (HRMS) analysis was performed using an HRMS ESI-TOF or ESI-MS  instrument. The fluorescent image of labeled bacteria was taken by Confocal Zeiss LSM 880 FAST AiryScan at the Biology Imaging Center at the Faculty of Science of Utrecht University.All NMR spectra were recorded at 400 MHz in Utrecht Institute for Pharmaceutical Sciences center with chemical shifts reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS). E. coli Rosetta BL21(DE3) pLysS transformed with pET28a-OleD-TDP16 plasmid, which was picked from the LB agar plate with 50 μg mL−1 kanamycin, was inoculated in 60 mL of LB with 50 μg mL−1 kanamycin at 37°C and in a shaker (200 rpm) overnight. The next morning, the starting culture was transferred into 6 L of LB medium supplemented with 50 μg mL−1 kanamycin and further grown in an incubator with shaking (200 rpm) at 37°C until the OD600nm reached around 0.6. IPTG was added into the medium cultures to bring the final concentration of IPTG to 0.4 mM and the cultures were incubated at 25°C for around 16 h with shaking at 200 rpm. Bacteria were concentrated further by centrifugation at 6000g and 4°C for 15 min. The cell pellet was subsequently resuspended in 100 mL of lysis buffer  and lysed with a cell disruptor (Constant systems) for 5 cycles while cooling at 4°C. After the lysis, the disrupted bacteria cells were incubated on ice for a further 20 min to degrade peptidoglycan, DNA, and RNA. Cell debris was removed by centrifugation  and then the supernatant was applied to 6 mL of nickel resin equilibrated in wash buffer . The protein was incubated with the beads for 2 h on a digital tube roller at 4°C. Then the resin was applied to a column followed by a washing step with 150 mL of chilled wash buffer. Subsequently, the protein was eluted with 10 mL of elution buffer . Purified protein was concentrated and desalted using a PD-10 desalting column , equilibrated with 50 mM Tris-HCl (pH 8.0), to provide 3 mL of the desired protein. The purity of the enzyme was determined by 10% SDS-PAGE, indicating the purity of more than 95%. The purified enzyme was stored at −80°C until use.We utilized a protocol based upon the previously published methods for the OleD enzyme . A singl4 (25 mM), and OleD enzyme (125 μM). The reaction was monitored by TLC . Besides, the color of the reaction mixture turned yellowish from almost colorless as the reaction progressed. After 24 h, the reaction was stopped by adding 1 mL chloroform to precipitate the OleD enzyme. The reaction medium was centrifuged at 2,000g to remove the OleD enzyme. The aqueous upper phase was carefully collected and transferred into a new tube, followed by lyophilization. The dried yellowish residue was dissolved in 0.25 mL of Milli-Q water and the desired product was purified by silica gel column using n-butanol: acetic acid: water (2:1:1) as eluent. The product  was concentrated on a vacuum rotary evaporator and the residue was lyophilized to get a white powder .Reactions were conducted at 37°C in 20 mL Tris-HCl buffer  with GlcNAz-CNP (5 mM), UDP (5 mM), MgSO1H NMR  δ 7.81 , 5.94 – 5.73 , 5.52 – 5.31 , 4.27 – 4.19 , 4.16 – 3.89 , 3.74 , 3.52 – 3.38 , 3.24 – 3.19 .13C NMR  δ 170.85, 166.40, 151.88,141.56, 102.56, 94.26, 88.60, 82.96 ,73.65, 72.99, 70.70, 69.45 , 65.01, 60.24, 53.63 , 51.52, 50,45.31P NMR  δ −11.44 , −13.20 .17H25N6O17P2 (M-H) 647.0928, found 647.0933.HRMS (ESI-TOF) m/z calcd for CMicrococcus flavus DSM 1790 or Staphylococcus simulans 22 bacteria was inoculated in 6 × 25 mL of CASO broth and grown overnight at 37°C in a shaker. The next morning, the culture was transferred into 4 L of fresh CASO medium, grown until an OD600nm = 1.0∼1.2 was reached, and harvested by centrifugation at 3,800 g at 4°C for 30 min. The supernatants were discarded, and bacteria were washed in Tris-HCl . The bacteria were resuspended in 50 mL per gram of wet weight in Tris-HCl buffer . A freshly prepared solution of DNAse and RNAse (both at 0.6 mg per gram of wet weight) in Milli-Q water was added as well as a solution of lysozyme in Milli-Q water (20 mg per gram of wet weight). The cells were then disrupted by 10 cycles in a cell disruptor (2.5 kPa). The resulting suspension was centrifuged at 4,000g and the supernatant was collected. The membrane fraction was obtained by centrifugation of the supernatant at 45,000g. The membranes were resuspended into 5 mL of Tris-HCl buffer  and frozen at −20°C until further use.A single colony of P) stock solution in acetone was dried under a nitrogen flow and mixed with 50 μL of UDP-GlcNAz (3 mM), 5 μL of Triton X-100 (10%), 30 μL of the membrane isolation, 5 μL of MgCl2 (0.15 M), 15 μL of Tris-Cl , and 30 μL of UDP-MurNAc-pentapeptide (UDP-Mpp) extract. The mixture was incubated at room temperature overnight. The reaction was stopped by adding 180 μL of butanol/6 M pyridine-acetate acid . The sample was thoroughly mixed followed by brief centrifugation to separate the different layers. The butanol layer was collected, washed with 100 μL of water, diluted with 400 μL of methanol, and loaded onto a DEAE-cellulose column . The column was rinsed with methanol (600 μL) and eluted with 0 mM, 100 mM, 300 mM, and 500 mM NH4HCO3 in the water fraction of chloroform/methanol/water (2:3:1), 600 μL for each concentration of eluent. 100 μL of each fraction was collected, dried, and dissolved in chloroform/methanol (2:1), analyzed by TLC with an eluent of chloroform/methanol/water/ammonium hydroxide (88:48:10:1), and stained by iodine. The fractions containing GlcNAz-Lipid II were lyophilized to give 75% yield.30 μL of a 1 mM undecaprenyl phosphate  and tris-hydroxypropyltriazolylmethylamine (THPTA) (Sigma-Aldrich)  were mixed in Tris-HCl buffer  and stirred at room temperature for 20 min. Sodium ascorbate  was added to the reaction mixture and the mixture was immediately dried under nitrogen flow. Subsequently, the residue was dissolved in Tris-HCl buffer  with 0.1% Triton X-100 and transferred into a tube containing a premixed solution of GlcNAz-labeled Lipid II  and dansyl alkene  to bring final concentrations of CuSO4 (0.1 mM), THPTA (0.5 mM), sodium ascorbate (2.5 mM), GlcNAz-DAP-Lipid II (0.1 mM) and dansyl alkyne (0.3 mM). The total volume of the reaction is 1.5 mL. The reaction was protected from the light and stirred at room temperature for 2 h, followed by extraction of the Lipid II by 1.8 mL of butanol/6 M pyridine-acetate . The butanol layer was washed with 1 mL water, collected, diluted with 4 volumes of methanol, and loaded onto a DEAE-Cellulose column . The column was, rinsed with 6 volumes of methanol, and eluted with 5 mL buffers at each step solvent gradient of chloroform/methanol/water (2:3:1), chloroform/methanol/100 mM ammonium bicarbonate (2:3:1), chloroform/methanol/200 mM ammonium bicarbonate (2:3:1), chloroform/methanol/300 mM ammonium bicarbonate (2:3:1), chloroform/methanol/500 mM ammonium bicarbonate (2:3:1), and to chloroform/methanol/1 M ammonium bicarbonate (2:3:1). The dansyl-Lipid II was present in the fraction of chloroform/methanol/1 M ammonium bicarbonate (2:3:1). The final product was obtained after lyophilizing the fractions . The neutral mass of dansyl-GlcNAz-Lipid II was determined as 2247.24 Da which is consistent with the calculated exact mass of 2246.15 Da.CuSOE. coli PBP1B, P. aeruginosa PBP1B, E. coli PBP1A, E. coli LpoB, P. aeruginosa LpoP, and E. coli LpoA were purified as previously described (references: E.c. PBP1B (Ec and PBP1BPa or 0.7 μM for PBP1AEc) in the presence or absence of activators (2 μM for LpoB and LpoP or 2.8 μM for LpoA) with 10 μM labeled substrate (dansyl-GlcNAz-Lipid II or dansyl-lysine-Lipid II) in 50 mM Hepes at pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05% TX-100. When indicated, reactions were supplemented with 10 μM unlabeled m-DAP-Lipid II, 50 μM moenomycin or 1 mM ampicillin. Reactions were incubated for 1 h (PBP1BEc and PBP1BPa) or 2 h (PBP1AEc) at 37 °C and then boiled for 5 min. Samples were vacuum-dried using a speed-vac desiccator and products were analyzed by Tris-Tricine SDS-PAGE as described previously . Reactieviously , with th−1 kanamycin) and were incubated until the OD600nm was about 0.6. 1 mL of cells were collected by centrifugation at 6,000g for 2 min BL21 (OleD) were resuspended in 200 μL of LB medium (with 50 μg mL−1 kanamycin). GlcNAz-CNP (0.2%) and 0.4 mM IPTG were added to the cell samples. Cells were incubated at 37°C for 2 h and then collected  and washed twice with 400 μL PBS buffer (pH 7.4). Subsequently, the cells were resuspended in PBS (pH 7.4) buffer and fixed at room temperature with 4% paraformaldehyde in PBS (pH 7.4) for 20 min. The cells were then washed twice with 200 μL PBS (pH 7.4). The cells were collected and redispersed in 25 μL of isopropanol: water (2:1) followed by the addition of 5 μL of CuSO4 solution (10 mM), 5 μL of THPTA (Sigma-Aldrich) (10 mM), 5 μL of freshly prepared sodium ascorbate (Sigma-Aldrich) (25 mM), and 10 μL of Alexa Fluor 488-alkyne (Sigma Aldrich) (100 μM). Cells were incubated at room temperature in a shaker for 30 min in the dark, subsequently washed 6 times with 200 μL of PBS (pH 7.4) and resuspended in 200 μL of PBS (pH 7.4) then prepared for imaging.Overnight pre-cultured BL21 cells harboring pET28a-OleD-TDP16 were inoculated into fresh LB medium (with 50 μg mL600nm = 0.06) and EDTA was added into the suspension to bring the concentration to 1.7 mM. The bacteria were incubated with EDTA for 20 min at room temperature. Afterward, the bacterial suspension was scanned by a fluorometer (excited at 488 nm and data recorded between 500 nm and 700 nm). Then, TMR-Vancomycin (0.8 μM) was added to the suspension that was incubated for 20 min at room temperature. The data of the emission spectrum was recorded between 500 nm and 700 nm. The data of the control group (0.8 μM TMR-vancomycin in PBS with 1.7 mM EDTA) was recorded at the same conditions and fluorometer settings as the other sample. The FRET signal was plotted by the subtraction of the emission spectrum of TMR-Vancomycin from the emission spectrum of the mixture of Alexa fluor 488 labeled BL21 (pET28a-OleD-TDP16) and TMR-Vancomycin, which was recorded between 500 nm and 700 nm and excited at 488 nm.BL21 (pET28a-OleD-TDP16) cells were grown in LB with GlcNAz-CNP (0.2%) and labeled with Alexa fluor 488 via click chemistry. The labeled bacteria were resuspended into 1 mL of PBS (ODBL21 pET28a-OleD-TDP16 cells were grown in 100 mL of LB medium in the presence or absence of 0.1% GlcNAz-CNP and the PG was isolated from these cells as previously published . PG was th volume 10% formic acid (aq) and placed in an autosampler vial. 2 μL of acidified muropeptide was injected onto a microbore RP-HPLC column  flowing at 15 μL min−1 delivered by a NanoAcquity UPLC system . The column temperature was 35°C. Buffer A was composed of water containing 0.1% (v/v) formic acid. Buffer B was acetonitrile containing 0.1% (v/v) formic acid. The following elution gradient was used: starting at 0% buffer B, rising to 1.3% B at 3 min, then on to 2.5% B at 20 min, rising to 6.0% B at 35 min, the gradient was ramped to 10% B at 45 min, then to 50% B at 50 min, followed by 2 min at 85% B, and finally 12.5 min re-equilibration at 0% B. The total run time was 65 min.LC/MS analysis was performed as recently described  with mod−1 and nebulizer pressure of 1.0 bar. MS data were acquired in positive ion mode over the range of 50∼2,000 m/z at a spectral rate of 2 Hz. MS/MS acquisition was performed on the top five intense precursors in each scan (ions excluded after two MS/MS events). The preferred charge state range was set at +1 to +4 (undetermined charge states were excluded) and the sampling rate varied from 0.5 Hz at low ion counts  to 5.0 Hz for high-intensity ion counts . The resulting MS spectral data was opened for analysis using Compass DataAnalysisTM software (Bruker).The HPLC column eluate was directed to the mass spectrometer  via an Apollo electrospray ion source (Bruker). The settings for the ion source were: capillary voltage and temperature settings of 3,200 V and 150 °C respectively, together with a dry gas flow of 6 L min1 as light-yellow solid .Glucosamine hydrochloric acid salt  was dissolved in methanol (150 mL). Sodium methoxide  was added into the solution drop by drop. The solution was stirred at room temperature for 20 min. Triethylamine  and chloroacetic anhydride  were subsequently added to the reaction solution. The reaction was kept stirring for up to 72 h, during which the reaction was monitored by TLC. When the starting material was consumed up, the reaction solution was filtered, and the filter residue was washed with methanol (50 mL). The filtrate was concentrated and dried to offer the residue. To a 250 mL round flask, the residue in acetyl chloride (75 mL) was added. The solution was kept stirring for 20 h at room temperature. The color of the reaction solution got dark from colorless. Then the solution was diluted by chloroform (400 mL), treated with the ice-water mixture (400 mL), washed with cold water (250 mL), and cold saturated sodium bicarbonate solution (3 × 400 mL). The organic layer was dried with magnesium sulfate and concentrated with a vacuum rotary evaporator. The brown residue was purified with silica gel column  with eluting  to afford compound 1H NMR  δ 6.86 , 6.19 , 5.38 , 5.20 , 4.47 , 4.29 , 4.27 – 4.25 , 4.11 , 4.05 – 3.94 , 2.08 – 2.03 .13C NMR  δ 171.11, 170.47, 169.11 , 166.45 (C8), 92.71 (C1), 70.92 (C5), 69.70 (C3), 66.84 (C4), 61.05 (C6), 53.88 (C2), 42.06 (C9), 20.62, 20.55, 20.49 .2 was obtained as an off-white solid .To a 50 mL round flask, compound 1  and acetonitrile (2.5 mL) were added. 2-chloro-4-nitrophenol  and trimethylamine (2.5 mL) was added subsequently. After the reaction was kept stirring at 40°C overnight, the reaction mixture was diluted with chloroform (10 mL), washed with sodium hydroxide solution (0.1 M) twice (2 × 5 mL), and washed with water (10 mL) before being dried by magnesium sulfate. After filtration, the filtrate was concentrated with a vacuum rotary evaporator and the resulting residue was purified by silica gel  column chromatography . Compound 1H NMR  δ 8.28 , 8.11 , 7.26 , 6.89 , 5.52 , 5.51 – 5.49 , 5.15 , 4.25 , 4.18 – 4.10 , 4.01 , 3.99 – 3.93 , 2.19 – 1.99 .13C NMR  δ 170.36, 170.35, 169.24 , 166.72 (C-10), 157.18 (C-14), 143.11 (C-17), 126.12 (C-18), 124.83 (C-19), 123.54 (C-16), 116.64 (C-15), 98.36 (C-1), 72.59 (C-5), 70.25 (C-3), 68.01 (C-4), 61.92 (C-6), 54.53 (C-1), 42.36 (C-11), 20.62, 20.58 .2  was dissolved in dimethylformamide in a 50 mL round flask with a stirring bar. Sodium azide  was added to the reaction mixture before the reaction was kept stirring for 6 h at 60°C. The reaction mixture was poured into ice water (20 mL) and extracted with ethyl acetate 3 times (3 × 20 mL). The organic phase was collected and washed with water 3 times (3 × 20 mL). The organic phase was washed with saturated sodium bicarbonate solution (10 mL) before being washed with water 3 times (3 × 20 mL) again and dried with magnesium sulfate. The organic phase was filtered, concentrated and the residue was purified by silica gel  column chromatography . Compound 3 was obtained as white solid .The compound 1H NMR  δ 8.25 , 8.08 , 7.25 , 6.79 , 5.56 – 5.53 , 5.53 – 5.48 , 5.14 , 4.24 , 4.14 , 4.00 – 3.93 , 3.92 , 2.06 – 2.05 .13C NMR  δ 170.43, 170.36, 169.25 , 167.36 (C-10), 157.21(C-14), 143.08 (C-17), 126.12 (C-18), 124.75 (C-19), 123.56 (C-16), 116.55 (C-15), 98.43 (C-1), 72.55 (C-5), 70.58 (C-3), 68.06 (C-4), 61.90 (C-6), 54.48 (C-2), 52.59 (C-11), 20.67, 20.62, 20.58 .3  in anhydrous methanol (30 mL) was added 25% (w/w) sodium methoxide . After the reaction was stirred overnight, the solution turned a little yellowish the next morning. Amberlite IRC-50 resin was washed with methanol and added to the reaction mixture, which was stirred until the pH of the mixture reached 5 to 6. The mixture was filtered by an Amberlite IRC-50 resin desalting column and concentrated, purified by silica gel column chromatography with eluent  to afford the off-white compound 4 .To compound 1H NMR  δ 8.31 – 8.25 , 8.17 , 7.45 , 5.20 , 3.84 , 3.77 , 3.72 , 3.53 – 3.51 , 3.50 – 3.44 , 3.26 .13C NMR  δ 166.58 (C-10), 157.06 (C-16), 141.12 (C-19), 124.70 (C-20), 123.56 (C-18), 121.89 (C-21), 115.44 (C-17), 98.49 (C-1), 76.90 (C-3), 72.73 (C-4), 69.13 (C-5), 59.71 (C-6), 54.37 (C-2), 50.35 (C-11).14H16ClN5O8Na] + ([M+Na] +) 440.05, found 440.06.ESI-MS m/z calcd for [CThe fluorescence intensity in the FRET experiments was measured by Cary Eclipse Fluorescence Spectrometer. The raw data was collected by Cary Eclipse Scan Application 1.1. The experiments were repeated at least twice. The figure was produced with GraphPad Prism version 8.0.2 from the raw data.


Page 1, abstract, line 14: “for ST11 CRKP” should read “for ST11 K. pneumoniae.”Volume 10, no. 5, e01690-22, 2022, P < 0.05)” should read “The median hospital stays of 30 CRKP-BSI patients who died during hospitalization and 116 CRKP-BSI patients who survived during hospitalization were 61.5 days (inter quartile range [IQR]: 78 days) and 47.5 days (IQR: 53 days). There were statistically significant differences in intensive care unit (ICU) admission and use of urinary catheters between the 2 groups (P < 0.05).”Page 2, lines 12 to 8 from bottom: “The median age of 30 CRKP-BSI patients died during hospitalization, and 116 CRKP-BSI patients who survived during hospitalization were 61.5 years old (Inter Quartile Range [IQR]: 78 years old) and 47.5 years old (IQR: 53 years old). There were statistically significant differences in intensive care unit (ICU) admission, hospital stay, and use of urinary catheters between the 2 groups (Page 3, Table 2, column 4, row 2: “0.036” should read “0.037.”Page 9, line 15 from bottom: “totaled 5.6 Mb” should read “averaged 5.6 Mb.”


Both products are formed via ring opening of the same intermediates, 1,3-dioxolan-2-ones, generated in situ from bromopropargylic alcohols and Cs2CO3.The reaction of bromopropargylic alcohols with phenols in the presence of Cs Of particular synthetic value is the addition to the triple bond of bromoacetylenes to provide vinyl adducts, which can undergo numerous transformations. For example, bromoacetylenes were demonstrated to add imidazoles, imidazolines furans, etc. furans by thermal heating of a dispersion of α-phenoxyketones in Al2O3. We involved the synthesized α-phenoxyketones 4 in this reaction. The results showed that instead of benzo[b]furan formation, α-ketol rearrangement of phenoxyketones 4a,f occurred to afford β-phenoxyketones 9a,b in 55–60% yields  [3, carminomycin I, epothilones) [We have shown that the main direction of the reaction of bromopropargylic alcohols and phenols in Css, etc.) –46 and ps, etc.) –49 and cs, etc.) , antideps, etc.) , amyloids, etc.) , urease s, etc.) , farnesys, etc.) –55, antihilones) –58 activ1Н and 13С NMR spectra were recorded on a Bruker DPX-400 spectrometer  in CDCl3 or (CD3)2CO using hexamethyldisiloxane as internal reference at 20–25 °C. IR spectra were measured on a Varian 3100 FT-IR Excalibur series instrument as thin films or KВr pellets. Microanalyses were performed on a Flash 2000 elemental analyzer. Melting points were determined using a Kofler micro hot stage apparatus. Mass spectra were recorded on a GCMS-QP5050A spectrometer made by Shimadzu Company. Chromatographic column parameters were as follows: SPBТМ-5, length 60 m, internal diameter 0.25 mm, thickness of stationary phase film 0.25 μm; injector temperature 250 °C, gas carrier – helium, flow rate 0.7 mL/min; detector temperature 250 °C; mass analyzer: quadrupole, electron ionization, electron energy: 70 eV, ion source temperature 200 °C; mass range 34–650 Da. The solvent was distilled DMF. Column chromatography was performed on silica gel 60 . Bromopropargylic alcohols 1a–c and chloropropargylic alcohol were prepared according to published methods [232a), naphthalen-1-ol (2b), naphthalen-2-ol (2c), 4-nitrophenol (2d), 2-nitrophenol (2e), p-cresol (2f), 4-methoxyphenol (2g), 4-bromophenol (2h), 4-allyl-2-methoxyphenol (2i) are commercial reagents. Commercially available starting materials were used without further purification. The structures of synthesized products have been proven by 1H, 13C and 2D  NMR techniques, as well as IR spectra. methods –2359. PhTypical procedure for preparation of phenoxyhydroxyketones 4 in DMF, 50–55 °C. To a stirred solution of Cs2CO3  and phenol  in DMF (5 mL) 4-bromo-2-methylbut-3-yn-2-ol  was added dropwise. The reaction mixture was stirred at 50–55 °C for 3 h, filtered and concentrated. The residue was purified by flash column chromatography on silica gel  to give products 3a , 4a , 5a  and 7 .File 1General information, synthetic procedures and additional optimization results, NMR spectra and characterization of synthesized compounds.