Atomically flat epitaxial (110) Mo films are grown on (0001) Al2O3. By impinging nitrogen ions, where the beam energy is fixed to 20 keV, we observe (111) γ-Mo2N diffraction and the formation of a γ-Mo2N layer from X-ray reflectivity. Magnetization and transport measurements clearly support a superconducting layer in the implanted film. Our strategy shows that formation of a buried superconducting layer can be achieved through ion implantation and self-annealing.Nitrogen ion implantation is a useful technique to put nitrogen ions into lattices. In this work, nitrogen ion implantation into epitaxial Mo films is performed to create a buried superconducting γ-Mo 2N, by low-energy nitrogen ion implantation in an epitaxial molybdenum thin film.Formation of chemically distinct interfaces, including crystalline buried-superconducting Mo Fractional boron doping into silicon using ion implantation enabled formation of the desired level of doping in semiconductors.3,4 In addition, for fabricating dilute magnetic semiconductors, magnetic ion implantation was used for room temperature ferromagnetism.2,5–7 Unusual luminescence and photo-activity in the implanted films were also reported.8–11 However, the implantation strategy is not limited to semiconductors. In recent, it was adopted for stabilizing meta-stable phase such as rare-earth-free permanent magnet such as Fe16N2 by implanting nitrogen ions into iron lattices.12 Thus, combining ion implantation technique in crystal synthesis may bring an another degree of freedom for tuning materials properties. In this regard, creation of a metal nitride from nitrogen ion implantation is important, since nitrogen molecules are normally very stable. So, it is not easy to decompose nitrogen molecules, incorporate nitrogen ions into the lattices, and form desired stoichiometry. Ones often used ammonia as a processing agent for nitridation.13–15 Even if ion implantation is rather destructive method to impinge small atomic or molecular nitrogen ions, usually N+ or N2+, into crystal lattices, it is expected to intercalate nitrogen ions effectively and stabilize them in the lattice through post-process: i.e. heat treatment. This leads to stabilize a highly-stabilized resistive surface and/or exotic physical properties like superconductivity, which will be introduced in this work.Ion implantation is a versatile technique to incorporate ions into crystalline lattices.16–21 It has been proven that ion beam implantation with wider nitrogen beam energy (up to 200 keV) and relatively high nitrogen ion dose (1016 ∼ 1017 ions cm−2) can induce the formation of γ-Mo2N, δ-MoN, and B1–MoN. It is generally known that higher ion incorporation could be possible, when lower energy and higher dose used.20 In this work, we observed evidence of buried superconducting-phase formation by ion implantation on (110) epitaxial Mo thin films using relatively low energy. First, we synthesized atomically flat (110) Mo thin films. The films were transferred to ion beam facility for atomic nitrogen ion (N+) beam implantation at low energy to minimize disordering of Mo atoms. The implanted films were tested to find potential formation of superconducting nitrides using X-ray scattering, cross-section transmission electron microscopy, atomic force microscopy (AFM), transport, and magnetization measurements.Nitridation of molybdenum using ion implantation is of considerable interest, since molybdenum nitrides can be mechanically strong and superconducting materials with different critical temperatures depending on nitrogen content.2O3 substrates  using custom-made DC magnetron sputtering. The detailed growth condition for Mo films are following: 5 mTorr as forming gas pressure (Pforming), 50 W of DC power, and 700 °C of substrate temperature (TS). X-ray diffraction  techniques such as X-ray reflectivity, 2θ–ω scan, φ-scan were employed to characterize structural information of epitaxial Mo films  results22 to estimate profile of implanted nitrogen ions and recoiled or disordered molybdenum ions. X-ray 2θ–ω scan and X-ray reflectometry (XRR) were specially adopted to see formation of buried molybdenum nitrides and destabilization of molybdenum by ion implantation. To observe microstructure and chemical inhomogeneity due to nitrogen ion implantation, cross-sectional transmission microscope  was used. Z-contrast imaging and energy dispersive X-ray spectroscopy (EDS) are performed on the highest nitrogen dosed sample (5 × 1016 ions cm−2 of N+). X-ray reflectivity fitting was performed with GenX software.22 After confirming the formation of the nitrides, we used SQUID magnetometer  and Physical Property Measurement System (Quantum design) to observe temperature dependence of magnetization and transport property. For a SQUID measurement, we used 100 Oe of magnetic field. For a transport measurement, we used conventional four probe transport geometry and the data is normalized by resistance value at T = 8 K .80 nm-thick epitaxial Mo thin films were grown on (0001) Alilms see . After c2O3. Clear Kiessig fringes show well-defined interfaces with about 80 nm in thickness. The obtained electron scattering length density (eSLD) of a Mo layer is 7.54 Å−2, which is very similar to the bulk value of Mo (eSLD = 7.64 Å−2). In addition, from X-ray reflectivity fitting, we also obtained the surface roughness information, which is about 1 nm. The value is similar to the value obtained from atomic force microscopy  Mo is stabilized on (0001) Al2O3. In addition to the determination of out-of-plane information, we performed off-axis φ scan to figure out complete epitaxial relationship. φ scans around (104) Al2O3 and (200) Mo were selected. We observed Al2O3 peaks with 120° apart, while we obtained six peaks from (200) Mo with 60° apart. Also, Mo peaks are 30° apart from the nearest Al2O3 peaks. It indicates potential texture in the plane. In 2O3 are shown with rectangular (110) Mo lattices. From the figure, it can be easily seen that [001] Mo does not coincide with [100] Al2O3. In addition, [001] Mo is at least 30° apart from [100] Al2O3.2O3, we performed nitrogen-ion implantation with 20 keV and various doses. A schematic diagram in θ angle as the dose increases. It indicates, by recoiling of Mo atoms, lattice expansion is taken place. Note that the significant lattice expansion was found, when the dose is above 1016 ions cm−2. In addition, when we checked rocking curve of (110) Mo, we observed its full width half maximum (FWHM) changes from 0.08° for as-grown Mo film to 0.14° for N+ implanted Mo film with 5 × 1016 ions cm−2. A new diffraction peak is shown when the dose reached 5 × 1016 ions cm−2. The new peak is located to that of (111) γ-Mo2N.20,23 The ion implantation experiments were performed by cooling the backplate of sample stage using chilled water, and the temperature of backplate is kept to 24 °C. It is likely that the temperature of Mo films during ion implantation is different from that of backplate potentially due to self-annealing during ion implantation,24–26 which will be a potential reason for formation of crystalline molybdenum nitride. From AFM results in Fig. S1,After confirming epitaxial synthesis of (110) Mo thin film on (0001) Al16 ions cm−2 of nitrogen ion dose, to estimate depth information of recoiled Mo and implanted nitrogen, TRIM was used to simulate distribution of nitrogen ions in the Mo film and distribution of recoiled Mo atoms  and (c)2N, which are well-matched with the simulation results. In addition, we clearly observed lattice expansion upon nitrogen implantation.After finding the formation new phase in 5 × 10Mo atoms . It is n2N from X-ray diffraction and chemical depth profile of the highly dosed sample, we performed X-ray reflectivity of N+ implanted Mo films. 16 ions cm−2, since it is expected to have the highest contrast due to high concentration of recoiled Mo and high dose of N+. After getting thickness parameters, we performed XRR fitting of other two samples, which are chemically less distinct. From XRR fitting of the film with 5 × 1016 ions cm−2, thicknesses of recoiled Mo layer, nitrogen implanted Mo layer, and unperturbed Mo layer are 6.64 nm, 19.31 nm, and 57.16 nm, respectively. The corresponding electron scattering length density (eSLD) of recoiled Mo layer, nitrogen implanted Mo layer, and unperturbed Mo layer are 5.87, 6.48, and 7.54 Å−2. Note that when comparing the eSLD values of the nitrogen-implanted Mo layer, the value is similar to that of γ-Mo2N within 3% of error.27After checking the formation of γ-Mo−2 for the case of 1015 ions cm−2, 6.60 Å−2 for the case of 1016 ions cm−2, and 5.87 Å−2 for the case of 5 × 1016 ions cm−2. It is rather drastic change above 1016 ions cm−2. However, eSLDs of nitrogen-implanted Mo layer are monotonically reduced by increase of dose: 7.45 Å−2 for the case of 1015 ions cm−2, 7.20 for the case of 1016 ions cm−2, and 6.48 for the case of 5 × 1016 ions cm−2. Lastly, we tracked roughness of each layer. Interestingly roughness of both nitrogen-implanted Mo layer and unperturbed Mo layer are high for cases of the lower ion dose, while for the case of 5 × 1016 ions cm−2, interfacial roughness significantly reduced. Note that the surface roughness of the ion-implanted films was significantly reduced. The results from XRR fitting is consistent with those of AFM in Fig. S1.†It confirms ion beam implantation creates three distinct layers see  as we sa2N layer is expected to be superconducting, we performed transport measurements and temperature dependent magnetization. First, 16 ions cm−2 shows clear diamagnetic signal below 5 K. However, other lower-dosed samples are not diamagnetic. The superconducting critical temperatures of γ-Mo2N from other groups are listed in 2N include solid state reaction,16 ion beam implantation,28 pulsed laser deposition,29 sputtering,30–34 ion beam assisted deposition,34 electron beam evaporation,20,35 plasma immersion ion implantation36,37 and post annealing28,36,37. However, in these papers, there was no information of superconducting critical temperatures, so it was not included in Tc is 5 K.21,23,38–42As a buried γ-Mo16 ions cm−2 shows superconducting transition at around 5 K. It's ascribed to creation of the γ-Mo2N layer through ion implantation. We found small residual resistance of our superconducting sample. The process may not form a perfect defect-free superconducting layer due to the nature of ion implantation. Note that we observed characteristic slope changes from transport results of the low fluence Mo films. This indicates the films are not superconducting at the given temperature ranges, but it is possible to see some difference in superconducting critical temperature at the lower than 1.8 K. Note that Tc of pure Mo is 1 K.43This feature of the superconducting zero resistance is also clearly seen in the temperature dependence of resistance in 2O3 by low energy nitrogen ion implantation. The realization of superconductivity is seen with 5 × 1016 ions cm−2 and 20 keV of atomic nitrogen ion beam. It was checked that structural changes were observed through ion implantation, and the new peak was determined to be (111) γ-Mo2N. We performed the model fitting with three-layer model, and through eSLD and layer tracking, we could trace the γ-Mo2N layer formed on epitaxial Mo.In conclusion, we observed clear a buried superconducting layer in an epitaxial (110) Mo film grown on (0001) AlThere are no conflicts to declare.RA-010-D0RA08533B-s001


Spongospora subterranea. Arguably, zoospore root attachment is the most critical phase of infection; however, the underlying mechanisms remain unknown. This study investigated the potential role of root-surface cell-wall polysaccharides and proteins in cultivars resistant/susceptible to zoospore attachment. We first compared the effects of enzymatic removal of root cell-wall proteins, N-linked glycans and polysaccharides on S. subterranea attachment. Subsequent analysis of peptides released by trypsin shaving (TS) of root segments identified 262 proteins that were differentially abundant between cultivars. These were enriched in root-surface-derived peptides but also included intracellular proteins, e.g., proteins associated with glutathione metabolism and lignin biosynthesis, which were more abundant in the resistant cultivar. Comparison with whole-root proteomic analysis of the same cultivars identified 226 proteins specific to the TS dataset, of which 188 were significantly different. Among these, the pathogen-defence-related cell-wall protein stem 28 kDa glycoprotein and two major latex proteins were significantly less abundant in the resistant cultivar. A further major latex protein was reduced in the resistant cultivar in both the TS and whole-root datasets. In contrast, three glutathione S-transferase proteins were more abundant in the resistant cultivar (TS-specific), while the protein glucan endo-1,3-beta-glucosidase was increased in both datasets. These results imply a particular role for major latex proteins and glucan endo-1,3-beta-glucosidase in regulating zoospore binding to potato roots and susceptibility to S. subterranea. For potato crops, host resistance is currently the most effective and sustainable tool to manage diseases caused by the plasmodiophorid  Spongospora subterranea f. sp. subterranea is a significant threat to sustainable potato production wherever potato crops are grown [S. subterranea develop powdery scabs that affect tuber quality and storage longevity, whilst root infection affects root function (absorption of water and nutrients) and can reduce tuber yields [S. subterranea diseases are very limited. In some cases, farmers may be able to select cultivars that are relatively resistant to S. subterranea based on market demands; nevertheless, no cultivar is immune to infection, and substantial disease can still result in varieties that are only moderately resistant. To date, host resistance to Spongospora diseases has been assessed in traditional glasshouse and field trials and, more recently, using a rapid and robust in vitro zoospore root-attachment bioassay [The plasmodiophorid biotrophic pathogen re grown . This sore grown ,2,3. Tubbioassay . S. subterranea zoospores discharge their contents into the plant cell walls via a particular ‘Rohr’ and ‘Stachel’ structure [Infection of plant hosts by zoospores is preceded by a distinct sequence of initial zoospore recognition and attachment. Pathogen reactions to a host can be modelled on this pattern, making it a promising target for disease prevention ,6,7. Foltructure ,8. Zoosptructure ,10. Our tructure . HoweverPythium spp. was found to be affected by different plant polysaccharides, whereas Phytophthora spp. zoospore root attachment varied with the presence of pectin, polyuronates and some inorganic cations [Previous studies on other pathosystems suggest that the molecular interactions between host-plant cell-wall surface components and the infective units of pathogens are critical in the management of pathogenesis and plant resistance ,12. The  cations ,22,23,24P. aphanidermatum to the root surfaces of cress (Lepidium sativum). They found that trypsin was effective in reducing the number of zoospore root attachments, as was root-surface mucus–polysaccharide modification with lectin and pectinase. Downer, Menge and Pond [P. cinnamomi in avocado roots. However, no study has yet characterized the biochemical basis of the interaction between plant roots and S. subterranea zoospore attachment.Enzymatic studies have been extensively used to examine zoospore–host interactions ,28,29,30Our previous research investigated the basis of host resistance to zoospore root attachment by analysing the whole-root proteins of resistant and susceptible cultivars using label-free proteomics  and diffS. subterranea sporosori samples were collected from powdery-scab-infected tubers of the potato cultivar ‘Kennebec’ from a commercial potato field in North-West Tasmania, Australia, 2020. Infected tubers were washed with tap water and left to air-dry in a cool and dark place for 1 to 2 d. The lesions from infected tubers were excised with a scalpel and then sifted through a 600 µm sieve. S. subterranea inoculum was stored at room temperature in the dark until use.3, 253 mg/L; Ca(NO3)2·4H2O, 722 mg/L; KH2PO4, 2.3 mg/L; MgSO4·7H2O, 120 mg/L; NH4NO3, 40 mg/L; Fe-EDTA, 20 mg/L; H3BO3, 140 µg/L; KCl, 400 µg/L; MnSO4·H2O, 63 µg/L; ZnSO4·7H2O, 115 µg/L; CuSO4·5H2O, 50 µg/L; and Na2MoO4·2H2O, 22 µg/L in deionized distilled water (DDW) [Zoospores were released by incubation of sporosori samples in Hoagland’s solution, which contained the following components: KNOer (DDW) . Aliquot2/s) at 22 °C. After one month, all plantlets were transferred into potato multiplication medium without the phytagel and grown for a further two weeks under a 16 h photoperiod using white fluorescent lamps (65 µmol/m2/s) at 22 °C.Tissue-cultured plantlets of the cultivars ‘Iwa’ and ‘Gladiator’ were further propagated in tissue culture in potato multiplication medium containing the following ingredients: 4.43 g/L of Murashige and Skoog (MS) salts, 30 g/L of sucrose, 0.5 g/L of casein hydrolysate, 0.04 g/L of ascorbic acid, 2.2 g/L of phytagel (pH 5.8) under a 16 h photoperiod using white fluorescent lamps  was dissolved in 100 µL of 50 mM ammonium bicarbonate buffer (pH 7.8) to achieve a concentration of 0.2 mg/mL. A vial of 50 units of proteomic-grade PNGase F  was dissolved in 100 µL of high-purity water to provide a concentration of 500 units/mL. A quantity of 1 mg of the cellulase solution was prepared  in 1 mL of 50 mM sodium acetate buffer (pH 5.0).For the PNGase F treatment, 45 µL of 50 mM ammonium bicarbonate buffer and 5 µL PNGase F solution  were added to each tube in group 1. Further, all three tubes were incubated at 37 °C for 1 h . For the cellulase treatment, 45 µL of 50 mM sodium acetate buffer and 5 µL of 1 mg/mL cellulase solution were added to each tube in group 1. Then, all tubes were incubated at 37 °C for 0.5 h . For the TS treatment, 45 µL of 50 mM ammonium bicarbonate buffer and 5 µL 0.2 mg/mL trypsin solution  were added to each tube in group 1, with 5 min of incubation at 37 °C . Then, the TS experiment was repeated with 15, 30, and 60 min incubations at 37 °C. After enzymatic treatment, all the processed root segments were assessed for in vitro zoospore root attachment using the method described below. Similarly, all root segments in group 2 (control) were assessed via in vitro zoospore root-attachment assays, directly without any pre-treatment. All root segments were assessed according to the in vitro zoospore root-attachment assay, as previously described . Root sen) for each batch [The zoospore root-attachment score for each cultivar/line in the screenings was normalized against the reference cultivars, ‘Gladiator’ and ‘Iwa’, with the first batch screening serving as a reference point (G1 + I1) to adjust for across-batch differences. The cultivar/line scores were further linearly scaled according to the reference-point correction coefficient (ɳch batch .(1)ɳn=GFollowing TS treatment, root samples for all incubation times  were prepared for proteomic analysis using C18 ZipTips , according to the manufacturer’s instructions. The samples were dehydrated through vacuum concentration and reconstituted in 12 µL HPLC loading buffer (2% acetonitrile and 0.05% trifluoroacetic acid in water). Thermo Scientific’s Ultimate 3000 nano RSLC system and Q-Exactive HF mass spectrometer, equipped with nanospray Flex ion source, were used to analyze peptides with nanoflow HPLC-MS/MS and Xcalibur software (ver 4.3). Three ml aliquots of each sample were initially pre-concentrated in an analytical 20 mm × 75 µm PepMap 100 C18 trapping column, followed by separation over a 60 m segmented gradient in a 250 mm × 75 µm PepMap 100 C18 analytical column kept at 45 °C, at a flow rate of 300 nL/m. The MS Tune software (version 2.9) parameters used for data acquisition were: 2.0 kV spray voltage, S-lens RF level of 60 and heated capillary set to 250 °C. MS1 spectra (390–1500 m/z) were acquired at a scan resolution of 60,000, followed by MS2 scans using a Top15 DDA method, with 20 s dynamic exclusion of fragmented peptides. MS2 spectra were acquired at a resolution of 15,000 using an AGC target of 2e5, a maximum IT of 28 ms and a normalized collision energy of 27.Solanum tuberosum UniProt reference proteome (UP000011115) comprising 53,106 entries. With the exception of the activation of the match-between-runs function, default parameters for mass error tolerances, missed trypsin cleavages, and fixed and variable modifications were used. The false-discovery rate was set to 0.01 for both peptide–spectrum matches and protein identifications. Protein intensity values were imported into Perseus software (version 1.6.15.0) for further analysis. Protein groups identified as potential contaminants and proteins only identified by site or by reverse database matching were removed, and LFQ intensity values were log2-transformed. The proteins were filtered to include only those detected in a minimum of eight samples, and remaining missing values were replaced with random intensity values for low-abundance proteins based on a normal distribution of protein abundances, using default Perseus parameters.Mass spectrometry raw files were processed using MaxQuant software (version 1.6.5.0), using the Andromeda search engine to search MS/MS spectra against the t-test comparison of all replicates (n = 12) of both resistant and susceptible cultivars, with a false-discovery rate (FDR) of 0.05 and an s0 value of 0.1 used to define significant proteins. The differentially abundant proteins were classified using the UniProt database (www.uniprot.org (accessed on 6 June 2020)), DAVID bioinformatics resources 6.8 (https://david.ncifcrf.gov/ (accessed on 1 March 2021)) and the KEGG database (www.genome.jp/kegg/ (accessed on 1 March 2021)).Differentially abundant proteins were identified using a p = 0.05). The TS time-course incubation study revealed that zoospore root attachment was comparable for all TS incubation times  using IBM SPSS Statistics 27. Zoospore root-attachment scores were analyzed using one-way ANOVA followed by protected Fisher’s LSD test to determine statistically significant differences at the 5% level  on zoospore attachment to potato roots, followed by a detailed proteomic analysis of the products of the trypsin shaving treatment .Zoospore root attachment was significantly reduced in root segments treated with trypsin and PNGase F when compared with the untreated control in both susceptible (‘Iwa’) and resistant (‘Gladiator’) cultivars . In contN-linked glycoproteins—in plant–pathogen interactions. To gain a better understanding of potential mediators, we used a TS approach, in which peptides were collected from ‘Iwa’ and ‘Gladiator’ roots after incubation in trypsin for 5, 15, 30 and 60 min to allow for the detection of proteins with different susceptibilities to trypsin digestion under non-denaturing conditions. Following mass spectrometry analysis of the TS samples, a total of 1235 proteins were identified, of which 979 were quantified across the 24 samples after filtering the data to exclude proteins detected in fewer than 8 samples  between the two cultivars. This analysis identified 262 DAPs, of which 132 and 130 proteins were found to be significantly higher or lower in abundance in ‘Gladiator’ compared to ‘Iwa’, respectively  is shown in n = 8 proteins), biosynthesis of nucleotide sugars (n = 7 proteins), and amino sugar and nucleotide sugar metabolism (n = 7 proteins). In contrast, proteins that were less abundant in the resistant cultivar were related to carbon metabolism (n = 12 proteins), carbon fixation in photosynthetic organisms (n = 8 proteins), glyoxylate and dicarboxylate metabolism (n = 6 proteins), and the pentose phosphate pathway (n = 5 proteins). Pathway analysis revealed alterations in metabolic pathways in both subsets of DAPs b. SpecifThe bioinformatic analysis of the complete set of DAPs identified by the TS experiment identified significant functional differences between the proteomes of resistant and susceptible cultivars. However, this included a high proportion of cellular components that may not be directly involved in facilitating attachment to root surfaces. Therefore, we used our previous proteomic dataset acquired from whole-root tissue analysis to filter the TS dataset , which eS-transferases (GSTs)  were significantly more abundant in the resistant cultivar specific to the TS experiment  in their transcript and protein levels were compared (S-transferases (GSTs) (M0ZQ26 and M0ZQ38) were among the proteins that were found at increased levels in both datasets.Of the 188 significant proteins that were unique to the TS dataset, 92 were more abundant in the resistant cultivar, while 96 were less abundant. Proteins that were detected at increased levels included globulin (M1C704), ER6 protein (M1AZC6) and B12D protein (M0ZLR3), while those that were reduced included wound-induced proteinase inhibitor 1 (P08454) and major latex proteins (M1CYU9 and M1BBE7) c. Of notperiment c. Fifty-compared d. Thirty2) for these proteins are compared in Further comparison of the TS dataset with the whole-root proteome analysis enabled us to identify proteins with consistently large changes in abundance in both datasets. We selected the 20 proteins with the greatest differences in abundance in the TS treatment (ten increased and ten reduced in resistant vs. susceptible cultivars), of which 17 were also identified in the whole-root proteomic dataset. The fold changes  from root surfaces resulted in a reduction in Pythiaceous zoospore attachment; treatments that block or remove terminal fucosyl residues were particularly effective [S. subterranea zoospores to the roots of two potato cultivars  by suppressing the expression of genes and transcription factors associated with defence and stress. Similar to this result, we showed that the resistant potato cultivar, ‘Gladiator’, had lower expression of major latex proteins than the susceptible cultivar ‘Iwa’.Comparison of proteins from the TS proteome study with the whole-root proteins revealed 188 DAPs that were significantly abundant in the TS treatment b. Major  just 14 ,58. Majo just 14 ,60. He,  just 14  revealedN-glycoproteins can suppress the zoospore root attachment considered in this study. Together with these results, the cell-wall stem 28 kDa glycoprotein is an interesting candidate for direct association with the susceptibility of potato roots to zoospore root attachment.The cell-wall stem 28 kDa glycoprotein c was anoS. subterranea infection. The abscisic acid- and environmental-stress-inducible protein (M0ZVK4) was one of the proteins that was differentially changed between the TS and whole-root protein analysis. This protein decreased in the TS treatment but increased in the whole-root protein analysis for the resistant cultivar. Abscisic acid is essential for numerous cellular processes, including seed development, germination, crop growth and root architecture mediation [Glycan endo-1,3-beta-glucosidase (P52401) was the protein with the largest fold change, being identified as significantly more abundant in the resistant cultivar with the TS treatment, and this finding is consistent with the whole-root protein analysis . Glucan ediation ,67. AccoS. subterranea zoospore attachment. From the in vitro zoospore root-attachment study, the enzymes trypsin and PNGase F were found to significantly reduce zoospore root attachment, whilst cellulase had no effect on zoospore root attachment. Our detailed proteomic analysis revealed broad-scale differences of root proteins between susceptible and resistant potato cultivars. These proteins within potato roots provide new insights into host resistance to zoospore root attachment at a proteomic level. Overall, this study provides an initial understanding of the biochemical and molecular bases of potato resistance to zoospore root attachment and is important for developing novel approaches in future disease management.This is the first report of an investigation of the biochemical basis of potato root-surface components in relation to S. subterranea zoospore root attachment, but there are some limitations that ought to be mentioned. Firstly, the TS peptide analysis identified a large number of intracellular proteins, which may have hindered the identification of lower-abundance cell-surface proteins. While different time points were assessed in this study, further refinement of the TS approach may help to minimize the background of intracellular proteins. Secondly, trypsin shaving is inherently a peptide-centric approach that cannot easily distinguish between different proteoforms and therefore may underestimate proteome complexity. However, future studies using glycoproteomics may lead to a better understanding of the role of protein glycosylation in cultivar resistance to zoospore root attachment. This work contributes to knowledge of the biochemical and molecular bases of


The disruption of blood-brain barrier (BBB), predominantly made up by brain microvascular endothelial cells (BMECs), is one of the characteristics of Alzheimer’s disease (AD). Thus, improving BMEC function may be beneficial for AD treatment. Tanshinone IIA (Tan IIA) has been proved to ameliorate the cognitive dysfunction of AD. Herein, we explored how Tan IIA affected the function of BMECs in AD.1–42-treated brain-derived endothelium cells.3 (bEnd.3 cells) was employed for in vitro experiments. And we performed molecular docking and qPCR to determine the targeting molecule of Tan IIA on Sirtuins family. The APPswe/PSdE9 (APP/PS1) mice were applied to perform the in vivo experiments. Following the behavioral tests, protein expression was determined through western blot and immunofluorescence. The activities of oxidative stress-related enzymes were analyzed by biochemically kits. Nissl staining and thioflavin T staining were conducted to reflect the neurodegeneration and Aβ deposition respectively.AβMolecular docking and qPCR results showed that Tan IIA mainly acted on Sirtuin1 (SIRT1) in Sirtuins family. The inhibitor of SIRT1 (EX527) was employed to further substantiate that Tan IIA could attenuate SIRT1-mediated endoplasmic reticulum stress (ER stress) in BMECs. Behavioral tests suggested that Tan IIA could improve the cognitive deficits in APP/PS1 mice. Tan IIA administration increased SIRT1 expression and alleviated ER stress in APP/PS1 mice. In addition, LRP1 expression was increased and RAGE expression was decreased after Tan IIA administration in both animals and cells.Tan IIA could promote Aβ transportation by alleviating SIRT1-mediated ER stress in BMECs, which ameliorated cognitive deficits in APP/PS1 mice.The online version contains supplementary material available at 10.1186/s12967-023-03889-y. As a progressive neurodegenerative disease in the 21st century, Alzheimer’s disease (AD) has become one of the greatest threats to human health . Brain mSalvia miltiorrhiza, has a strong pharmacological effect on blood vessels. Tan IIA plays a therapeutic role in atherosclerosis, myocardial ischemia, coronary heart disease, and cerebral ischemia [Tanshinone IIA (Tan IIA), the representative of liposoluble components of ischemia . In addiischemia . What’s ischemia . However+/NADH ratio. Sirtuins is essential for DNA repair, anti-inflammation, and anti-oxidation [Sirtuins belong to class III histone deacetylase (HDAC), whose catalytic activity depends on the dynamic change of NADxidation . In mammxidation . Decreasxidation . Moreovexidation . SIRT1 cxidation . Endoplaxidation . ER strexidation . And ER xidation . Additioxidation . Whether1-42-treated brain-derived endothelium cells0.3 (bEnd0.3 cells) and APPswe/PSdE9 (APP/PS1) double transgenic mice were employed to unravel the role of Tan IIA in BMECs function and cognitive deficit. Tan IIA postponed the neurodegeneration, and significantly reversed cognitive dysfunction in AD. Tan IIA accelerated Aβ transportation in BMECs through relieving SIRT1-mediated ER stress.In the present study, Aβ2 contained in the atmosphere. Once cells had fused to 80–90%, they were treated with 0.25% EDTA for 1.5 min. Complete medium was then added to stop the digestive process. Cells were centrifuged, then the supernatant was discarded, and fresh complete medium was added to re-suspend.bEnd0.3 cell line was obtained from Jennio Biological Technology Co., Ltd. and incubated by the medium contained 45 mL DMEM (Gibco Invitrogen Corporation), 5 mL FBS (Gibco Invitrogen Corporation), 50 U penicillin, and 50 µg streptomycin at 37 °C with 5% CO1–42. After dissolving Tan IIA with dimethyl sulfoxide , it was diluted to the appropriate concentrations with DMEM without FBS. And Aβ1–42 (GL Biochem Ltd.) was treated with Hexafluoroisopropanol  for 1 h at room temperature, fully dissoluted by DMSO, cultivated at 4 °C for 24 h, and then diluted to different concentrations with DMEM.Subsequently, the cells were placed in 96-well plates, and administered with different concentration of Tan IIA . Following the pretreatment with Tan IIA for 2 h, cells were incubated with Aβ1–42 for another 24 h. bEnd0.3 cells were finally divided into four groups: control, Aβ1-42 10 µM, Aβ1-42 10 µM + Tan IIA 20 µM, Aβ1–42 10 µM + Tan IIA 20 µM + EX527 10 µM.The MTT test was used to determine cell viability for establishing the appropriate concentration and time of Tan IIA and AβTo screen which of the Sirtuins family binds most closely to Tan IIA, molecular docking was used for the preliminary prediction. To begin with, the PDB format files of SIRT1-7 were retrieved and downloaded from RCSB PDB database. Then, these files were processed by AutoDockTools 1.5.6 for dehydration, adding polarity hydrogenation and Gasteiger charges, and saved in PDBQT format. Secondly, the PubChem database was employed to obtain the 3D structure of Tan IIA. Thirdly, the AutoDockTools was employed to create the grid box with the size and coordinates recorded. Afterwards, both the molecular docking was performed and binding energy was calculated by Autodock Vina 1.1.2. Finally, the interaction between SIRT1-7 and Tan IIA were visualized by PyMOL 2.4.0.−ΔΔCt method was used to calculate the relative expression levels of each gene. Table A total RNA extract was prepared from bEnd0.3 cells using Trizol reagent. The concentration of RNA was then measured by NanoPhotometer (Implen). Evo M-MLV RT Kit (Accurate Biology) was employed to remove the genomic DNA and perform the reverse transcription. PCR amplification was done using a SYBR green kit (Accurate Biology). After amplification, the melting curve and amplification curve were exported, while the 2 The Nanjing University provided APP/PS1 transgenic mice, as well as transgenic negative mice of the same sex and age. At six months old, all mice were then randomly assigned to 4 groups (each with ten mice): WT group, APP/PS1 group, Tan-L group, and Tan-H group. Mice in WT group and APP/PS1 group were administrated by 0.9% saline, while mice in Tan-L group and Tan-H group were treated by Tan IIA at doses of 10 and 20 mg/kg/d. Tan IIA was dissolved in 0.2% DMSO and diluted with 0.9% saline before administrated to the animals. All mice were fed through gavage once a day for 8 weeks. The method of administration to the animals were determined with reference to the previously described protocol . In all We performed the open field experiment according to our previous study , 20. TheThe APP/PS1 transgenic mice also underwent a novel object recognition test to evaluate their episodic memory ability. We performed the novel object recognition test according to our previous study , 22. To Based on Morris’s approach, the Morris water maze test measures the memory and learning abilities of APP/PS1 mice . The Mor18 F-FDG PET/CT scanning. And food and water were withheld from the experimental animals for 6 h prior to the anesthesia. Then the anesthesia was achieved by inhaling pure oxygen mixed with 1.5% isoflurane during the experiment. Next, the radioactive tracer 18 F-FDG was injected into the tail vein of mice after full anesthesia. After 1 h of 18 F-FDG injection in mice, 10 min-PET images were acquired and statistically evaluated.The blood glucose levels of each mouse were monitored before the PET/CT imaging. When the blood glucose level was in the normal range (7.0–10.1 mmol/L), the mouse was approved to perform the GSH-PX, CAT, and T-SOD are 3 kinds of antioxidant enzymes, and MDA is a lipid peroxide degradation product. One of the distinguishing features of AD is oxidative stress, characterized by a low activity of antioxidant  and high level of MDA production. These oxidative stress markers can reflect the free radical content in tissues. In this study, The Nanjing Jiancheng Bioengineering Institute provided the detection kits for CAT, MDA, T-SOD, and GSH-PX. The supernatant was obtained after homogenizing and centrifuging the samples to determine the protein concentration by the NanoPhotometer. A series of measurements were carried out in line with the manufacturer’s instructions to determine the level of MDA, CAT, T-SOD, and GSH-PX. Finally, the absorbance was determined by Universal Microplate Spectrophotometer (Bio-Rad).RIPA buffer containing proteinase and phosphatase inhibitors was utilized for homogenizing and lysing bEnd0.3 cells and the cortex tissues, followed by SDS-PAGE with equal amounts of protein separated. Transferring the proteins to PVDF membranes (Millipore) and then blocking them in 5% skim milk for 70 min at room temperature were followed by incubation of the membranes through adding primary antibodies overnight at 4 °C and subsequent secondary antibodies for 90 min at room temperature. Routinely, protein load was determined by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and quantified by NIH software Image J.Paraffin-embedded perfused brain tissues were cut into sections of 5-µm thickness for Nissl staining. After sections had been dewaxed and rehydrated with xylene, ethanol, and distilled water, they were immersed into Nissl stain (Nanjing Jiancheng Bioengineering Institute) for 10 min at room temperature. Subsequently, the slides were washed by distilled water, dehydrated by ethanol, transparentized by xylene, sealed with neutral balsam, and eventually viewed by a light microscope (Leica Microsystems). For the analysis of hippocampal CA2 and cortex regions, Image J was used to count the number of Nissl bodies, and GraphPad Prism was used for the statistical analysis.6 per well, treated with Tan IIA, Aβ1 − 42, and EX527. After that, the cells were fixed with 4% paraformaldehyde (Aladdin) for 20 min at room temperature. In the next step, we permeabilized the cells and sections with 0.5% Triton X-100 (Solarbio) at 37 °C for 15 min, and then blocked them with 5% Bovine Serum Albumin  at 37 °C for 30 min, and subsequently incubated them with primary antibodies and secondary antibodies for the same temperature and time as western blot. After being sealed by antifade mounting medium with DAPI (Beyotime), images were obtained under a Leica Microsystems light microscope and analyzed by Image J software (n = 3).Brain paraffin sections were prepared by embedding the perfused brain tissues in paraffin and then cutting it into 5-µm thick sections for indirect immunofluorescence. After being dewaxed and rehydrated by xylene, ethanol, and distilled water, the slides were heated by microwave in sodium citrate buffer (Biosharp) for antigen retrieval. Cellular immunofluorescence was performed in 24-well plates, with bEnd0.3 cells seeded at 0.25 × 10The brain paraffin sections were dewaxed with xylene, rehydrated with ethanol and distilled water, and washed three times by PBS for 5 min. Then, each 5-µm thick section was incubated with 80 µL Thioflavin T mixture (1 mg Thioflavin T dissolved in 100 µL PBS) for 10 min. Next, the slides were washed by PBS, dehydrated by ethanol, sealed with neutral balsam, and eventually analyzed by fluorescence microscope (Leica Microsystems). Notably, Thioflavin T staining was performed in the cortex and hippocampus of the mice, with three images from three mice analyzed for each treatment.P< 0.05.GraphPad Prism 8.3.0 software (GraphPad) and SPSS 25.0 statistical software (IBM) were employed to perform all statistical analysis. All values were presented in the form of mean ± standard deviation (SD). when comparing differences among more than two groups, one-way ANOVA and two-way ANOVA were applied, followed by Tukey HSD test for multiple comparisons. One-way ANOVA was used for one independent variable, while two-way ANOVA was performed for two independent variables. It was determined that statistical significance was defined as 1–42 was treated to bEnd0.3 cells with concentrations of 1, 5, 10, and 20 µM for 24 h, and the cell survival rate reached to 93.9%, 78.6%, 51.2%, 45.8% , P-PERK (phosphorylate PKR-like ER), P-IRE-1α (phosphorylate inositol-requiring enzyme), P-eIF-2α (phosphorylate initiation factor 2), XBP1 (x-box binding protein 1), CHOP (C/EBP homologous protein) and a decrease of PDI (protein disulfide isomerase) in Aβ group as compared to the control group. Tan IIA reversed the expressions of these proteins, while EX527 blocked this effect  to correct the misfolding of ER lumen and dysregulation of calcium homeostasis . In the 22 cells . Above a22 cells . Also, i22 cells . Our stu2+ homeostasis, and reducing ATP production, giving rise to neuronal apoptosis [Oxidative stress, the common denominator of neurodegenerative diseases, produces free radicals that can cause oxidative damage to Aβ itself, and then lead to the deposition of Aβ, which is closely related to the pathogenesis of AD . Converspoptosis . In addipoptosis –53. Furtpoptosis . Oxidatipoptosis , 56. In Salvia miltiorrhiza, drive it to be a promising drug for treating AD.Taken together, our study elucidated that Tan IIA could reverse learning and memory decline by facilitating Aβ transportation, and its possible mechanism was to relieve SIRT1-mediated ER stress in the brain microvascular endothelium cells. However, the detailed mechanism behind SIRT1 mediating ER stress (such as whether its deacetylase function contributes to this) remains to be elucidated. Additionally, the neuroprotective effect of Tan IIA on AD still needs to be confirmed in clinical trials. The pharmacological properties of Tan IIA, which is derived from Additional file 1. Additional figures and table.


Alzheimer’s disease (AD) is the most common form of dementia. Although previous studies have selectively investigated the localization of amyloid-beta (Aβ) deposition in certain brain regions, a comprehensive characterization of the rostro-caudal distribution of Aβ plaques in the brain and their inter-regional correlation remain unexplored. Our results demonstrated remarkable working and spatial memory deficits in 9-month-old 5xFAD mice compared to wildtype mice. High Aβ plaque load was detected in the somatosensory cortex, piriform cortex, thalamus, and dorsal/ventral hippocampus; moderate levels of Aβ plaques were observed in the motor cortex, orbital cortex, visual cortex, and retrosplenial dysgranular cortex; and low levels of Aβ plaques were located in the amygdala, and the cerebellum; but no Aβ plaques were found in the hypothalamus, raphe nuclei, vestibular nucleus, and cuneate nucleus. Interestingly, the deposition of Aβ plaques was positively associated with brain inter-regions including the prefrontal cortex, somatosensory cortex, medial amygdala, thalamus, and the hippocampus. In conclusion, this study provides a comprehensive morphological profile of Aβ deposition in the brain and its inter-regional correlation. This suggests an association between Aβ plaque deposition and specific brain regions in AD pathogenesis. Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta (Aβ) plaques and the aggregation of neurofibrillary tangles (NFTs) caused by the hyperphosphorylated tau protein, and involves mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1), presenilin 2 (PSEN2), and apolipoprotein E (APOE) . PatientSeveral transgenic models have been developed to overexpress mutant forms of the amyloid precursor protein to mimic AD pathologies such as Aβ deposition. The transgenic 5xFAD mouse model was developed in 2006 to study late-onset AD. This mouse model consists of five familial AD mutations: Swedish APP mutation (K670N/M671L), Florida APP mutation (I716V), and London APP mutation (V717I), PSEN1 M146L mutation, and the PSEN1 L286V mutation . These mn = 10) and C57bL/6J wildtype (WT) mice (n = 9) were socially housed in standard cages (4–5 mice per cage) with access of food and water ad libitum. The female 5xFAD mouse model was selected based on the sex-biased epidemiological profile and neuropathological development in women compared to men  and 4% paraformaldehyde. Brain samples were harvested and post-fixed for 24 h in 4% paraformaldehyde, followed by incubation in 15 and 30% sucrose solutions until brains sank to the bottom. The brains were snap-frozen in liquid nitrogen before storing at −802O2 in 0.01 M phosphate buffered saline and 0.5% Triton X-100 (PBS-T) for 15 min, and then incubated in 1% bovine serum albumin for 15 min. A primary mouse anti-human 4G8 antibody  was added and sections were incubated at 4oC for 24 h. After rinsing, secondary biotinylated goat anti-mouse antibody  was added and sections were incubated at room temperature for 90 min, followed by avidin and biotinylated horse radish peroxidase  and further incubated for 2 h. Finally, sections were incubated in 1 mg/mL 3,3′-diaminobenzidene tetrahydrochloride  in Tris–HCl with 0.005% H2O2 and 8% nickel ammonium sulphate. After dehydration, sections were mounted and cover-slipped with Permount . Color images were acquired under a Zeiss AxioPhot upright microscope  at 5× magnification.The immunohistochemistry procedures were performed as previously described with minor modifications . Brains n = 4–6 mice, around 12–48 sections) in each selected regions of interest (ROIs) were quantified using ImageJ  using the threshold analysis method. In this method, we first use the free hand selection tool to outline the entire region, then the image was divided into two classes of pixels, the foreground Aβ plaque and the background . The methodology of intensity evaluation was conducted as previously described . The evackground .p-value less than 0.05, corresponding to a network correlation coefficient higher than ± 0.9. The resulting 3D model was visualized in R using the cocoframer package  training latency was analyzed by factorial mixed design ANOVA for day 1–7. Inter-reliability analysis was performed to qualitatively assess the reliability of the intensity grading by the two researchers. Finally, Pearson correlation coefficients were calculated to examine the inter-regional correlation of the Aβ plaque deposition.All data analyses were performed in IBM SPSS Statistics 27. The results were reported as the mean ± S.D, unless otherwise indicated. Kolmogorov-Smirnov test was performed to examine data normality. The Y-maze and OFT results were analyzed by independent samples t(16) = 2.477, p = 0.025] and the frequency to enter the novel arm  in 5xFAD mice compared to the wildtype control (t(16) = 0.445, p = 0.662] between 5xFAD mice and wildtype mice  = 6.066, p < 0.001], group , and their interaction  (t(16–17) = < −2.452, p < 0.025], but no significant differences were observed on days 1, 3, and 6  compared to wildtype mice. In the MWM probe test for long-term memory function, there was a significant decrease in the frequency to enter the target quadrant in 5xFAD mice  compared to wildtype mice, but no significant differences were found for the other quadrants   compared to wildtype mice. We found no significant differences in the latency to escape in MWM probe test for short-term memory function  , barrel field (5.88 ± 1.3%), forelimb region (6.40 ± 2.21%), dysgranular zone, hindlimb region (8.76 ± 3.58%), jaw region, shoulder region, trunk region, upper lip region, and secondary region of the somatosensory cortex, and piriform cortex (6.94 ± 2.00%). We also observed moderate Aβ deposition in the primary motor cortex (3.42 ± 1.27%), secondary motor cortex (2.75 ± 0.71%), prelimbic cortex (1.86 ± 0.69%), infralimbic cortex (1.86 ± 1.26%), dorsal peduncular cortex of the medial prefrontal cortex, area 1 and area 2 of the cingulate cortex, dorsolateral, lateral (2.59 ± 0.83%) and ventral (2.52 ± 0.95%) regions of the orbital cortex, dorsal and ventral (2.38 ± 1.12%) and posterior regions of the agranular insular cortex (5.94 ± 0.9%), and intermediate part of the endopiriform claustrum. In contrast, only mild Aβ deposition was detected in the medial part of orbital cortex, dorsal and ventral regions of the endopiriform claustrum, and dorsal and ventral claustrum –4A.We observed moderate Aβ deposition in the subthalamic nucleus, nucleus accumbens shell, lateral accumbens shell, and zona incerta; whereas mild Aβ deposition was detected in the pars compacta and pars reticulata of substantia nigra, lateral substantia nigra, nucleus accumbens core, ventral pallidum, caudate putamen, globus pallidus, and internal capsule. However, we did not detect Aβ deposition in the dorsal and medial regions of the substantia nigra pars compacta, or external capsule –6B.We observed moderate Aβ deposition in the anterior cortical amygdaloid area, posterior basolateral amygdaloid nucleus, anterior basolateral amygdaloid nucleus (2.99 ± 0.68%), medial posterior amygdala (2.76 ± 0.98%), medial anterior amygdala (1.22 ± 0.24%), and amygdalopiriform transition area. We also detected mild Aβ deposition in the anterior amygdaloid area, posterior basomedial amygdaloid nucleus, anterior basolateral amygdaloid nucleus, capsular of central amygdaloid nucleus, extended amygdala, medial amygdala (1.82 ± 0.40%), lateral amygdaloid nucleus, and posteromedial cortical amygdaloid nucleus. However, we found no Aβ deposition in the lateral and medial central amygdaloid or bed nucleus of the stria terminalis –6B.We found high levels of Aβ burden in the thalamus of 5xFAD mice, particularly in the posterior thalamic nucleus (6.82 ± 2.78%), ventral posterolateral thalamic nucleus (7.84 ± 3.20%), ventral posteromedial thalamic nucleus (6.26 ± 2.20%), and ventrolateral thalamic nucleus. We observed moderate Aβ deposition in the submedius thalamic nucleus, lateral posterior thalamic nucleus, parafascicular thalamic nucleus, and posterior intralaminar thalamic nucleus. We also detected mild Aβ deposition in the rhomboid thalamic nucleus, mediodorsal thalamic nucleus, subparafascicular thalamic nucleus, and ventral anterior thalamic nucleus. However, we did not detect any Aβ deposition in the anterior and posterior paraventricular thalamic nuclei, paratenial thalamic nucleus, or the reuniens thalamic nucleus –5B.We did not detect Aβ deposition in the hypothalamus of 5xFAD mice, including the paraventricular hypothalamic nucleus, striohypothalamic nucleus, posterior, dorsal, ventromedial, dorsomedial, central, and ventrolateral regions, shell region, lateral hypothalamus, lateral tuberal region, ventrolateral part of the hypothalamic area, and medial tuberal nucleus –5B.In the dorsal hippocampus of 5xFAD mice, we found high Aβ deposition in dentate gyrus (5.16 ± 1.43%), while moderate Aβ deposition was observed in CA1 (5.56 ± 1.45%), and CA3 (3.69 ± 1.83%) subregions of the dorsal hippocampus. In the ventral hippocampus, high Aβ deposition was shown in dentate gyrus (6.54 ± 2.92%), CA1/2 (8.83 ± 2.76%), CA3 (9.06 ± 2.86%), dorsal, transition area (18.38 ± 0.93%), and ventral subiculum –6B.In 5xFAD mice, we detected high Aβ burden in the external cortex of the inferior colliculus (5.19 ± 1.08%), whereas moderate Aβ deposition was found in the pontine reticulotegmental nucleus, paranigral nucleus, and intermediate reticular nucleus. We also observed mild Aβ deposition in the dorsal cortex of inferior colliculus, P1, ventrolateral periaqueductal gray, ventral tegmental area, parainterfascicular nucleus, ventral tegmental decussation, lateral superior olive, dorsal nucleus, subnucleus B, and subnucleus C of medial nucleus of the inferior olive, lateral terminal nucleus, supragenual nucleus, and external and rotundus part of the cuneate nucleus. However, we did not detect Aβ deposition in several midbrain regions, including the superficial layer, optic nerve layer, brachium, zona layer, intermediate gray layer, intermediate white layer of superior colliculus, dorsal lateral, dorsal medial, and lateral areas of the periaqueductal gray, caudal, dorsal, interfascicular, lateral, and ventral dorsal raphe, median raphe, nucleus of the trapezoid body, gracile nucleus, gracile fasciculus, median accessory nucleus, and cuneate nucleus of the medulla –7B.In 5xFAD mice, we found high Aβ deposition in the dorsal, dorsolateral, and ventral intermediate entorhinal cortex, as well as binocular and monocular areas of the primary visual cortex. We also observed moderate Aβ deposition in the ectorhinal cortex, medial entorhinal cortex, A, B, and C regions of the retrosplenial granular cortex, retrosplenial dysgrandular cortex (2.3 ± 0.49%), primary visual cortex (2.68 ± 0.81%), lateral (2.7 ± 0.55%), mediolateral, and mediomedial (2.29 ± 0.60%) areas of the secondary visual cortex, primary auditory cortex (2.18 ± 0.66%), dorsal (1.99 ± 0.63%), and ventral (1.99 ± 0.53%) areas of the secondary auditory cortex, and the temporal association cortex (1.62 ± 0.37%) –7B.We observed mild Aβ deposition in the superior cerebellar peduncle, paraflocculus of the cerebellum, superior, and lateral vestibular nuclei of 5xFAD mice . HoweverIn 5xFAD mice, we detected moderate Aβ deposition in the gelatinous layer, interpolar, caudal, and oral areas of the spinal trigeminal nuclei, and anterior areas of the anterior commissure. We also observed mild Aβ deposition in the central cervical nucleus of the spinal cord, intrabulbar and posterior nerve of the anterior commissure, magnocellular nucleus of the posterior commissure, nucleus of the posterior commissure, and island of Calleja . We founWe examined the possible inter-regional correlation by determining the Aβ plaque burden in the ROIs and their inter-regional correlations in 36 regions. The correlations are presented in the correlation matrix in In this study, we showed 9-month-old adult 5xFAD mice had significantly impaired spatial learning and memory functions. These results are in line with the memory deficits found in human AD patients . As the The deposition of Aβ in the cerebral cortex in AD patients has been confirmed by many studies using polarization sensitive optical coherence microscopy (PS-OCM), positron emission tomography (PET), and the magnetic resonance imaging (MRI) . MoreoveIn the correlation matrix, the medial prefrontal cortex and somatosensory cortex showed strong correlations with the hippocampus. The cerebral cortex is highly involved in hippocampal-dependent memory formation, given that it receives numerous projections from the hippocampus. The projection from the hippocampus to medial prefrontal cortex regulates social, hippocampal-dependent and fear memory ,2021, whvia regulating olfactory information to hypothalamus . These regions also receive projections layer V of the cortex layer V of the cortex from the hippocampus and are involved in hippocampal memory systems . The VTAWe are first to show mild Aβ deposition in both the VTA and substantia nigra of 5xFAD mice, which suggests mild Aβ deposition may be sufficient to suppress the dopaminergic system and disrupt memory encoding and memory consolidation processes in 5xFAD mice, although the dopaminergic system could also be affected by other AD pathologies. Our study also demonstrated for the first time mild to moderate Aβ deposition in the nucleus accumbens of 5xFAD mice. The nucleus accumbens is a key regulator of reward and satisfaction, and also receives projections from the hippocampus to mediate decision-making processes  and impuWe found Aβ deposition in various regions of the thalamus of 5xFAD mice. Deposition of Aβ in thalamus was also reported in post-mortem human AD brain tissues and in AD models by positron emission tomography . The thaWe did not detect Aβ deposition in the hypothalamus of 5xFAD mice, which is contrary to the report of Aβ deposition in the paraventricular nucleus of the hypothalamus in autopsy brain of AD patients . The hypAlthough we observed Aβ deposition throughout the olfactory areas except for the olfactory nerve, the effect of the Aβ deposition on olfactory memory was ambiguous. Two studies suggested that 5xFAD mice retain intact olfactory memory from 3 to 15 months of age without olfactory impairment , althougWe also did not detect Aβ deposition in the periaqueductal gray of 5xFAD mice, which contradicts the findings in a post-mortem AD brain study that observed senile plaques in the periaqueductal gray in 81% of samples . The perIn summary, the present findings provide comprehensive insights on the rostral-caudal distribution profile of Aβ depositions and their inter-regional correlation in adult 5xFAD mouse brain. Although our study focused on Aβ deposition, other hallmarks of AD  are equally important and will need to be investigated to further enhance translation from animal models to clinical applications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong (No. 4807-18).TS, YS, MLF, and LWL: conceptualization, funding acquisition, project administration, resources, and supervision. KCT, JR, SCC, and LS: data curation and formal analysis. KCT, JR, SCC, KHW, LS, CHP, YW, TS, LA, RCCC, YS, MLF, and LWL: investigation. KCT, JR, SCC, YS, MLF, and LWL: methodology. KCT, JR, SCC, LA, and KHW: visualization and validation. KCT and LWL: writing–original draft. JR, SCC, KHW, LS, CHP, YW, TS, LA, RCCC, YS, and MLF: writing–review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The use of microfragmented adipose tissue (µFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis (OA), is gaining popularity, following positive results reported in recent case series and clinical trials. Although these outcomes were postulated to rely on paracrine signals, to date, a thorough fingerprint of released molecules is largely missing. The purpose of this study was to first characterize both structure and cell content of unprocessed lipoaspirate (LA) and µFAT, and further identify and frame the array of signaling factors in the context of OA disease, by means of high throughput qRT-PCR for extracellular-vesicle (EV) embedded miRNAs and proteomics for tissue and secreted factors. Cell count showed reduction of blood cells in µFAT, confirmed by histological and flow cytometry analyses, that also showed a conserved presence of structural, endothelial and stromal components and pericytes. In the secretome, 376 and 381 EV-miRNAs in LA and µFAT, respectively, were identified. In particular, most abundant and µFAT upregulated EV-miRNAs were mainly recapitulating those already reported as ASC-EVs-specific, with crucial roles in cartilage protection and M2 macrophage polarization, while only a scarce presence of those related to blood cells emerged. Furthermore, secretome proteomic analysis revealed reduction in µFAT of acute phase factors driving OA progression. Taken together, these results suggest that processing of LA into µFAT allows for removal of blood elements and maintenance of tissue structure and stromal cell populations, and possibly the increase of OA-protective molecular features. Thus, microfragmentation represents a safe and efficient method for the application of adipose tissue properties in the frame of musculoskeletal disorders. Osteoarthritis (OA) is one of the most widespread causes of disability worldwide, affecting 7% of the global population and responsible for 2% of the total global years lived with disability . OA has In this category, platelet-rich plasma (PRP)- and mesenchymal stromal cell (MSC)-based products have rapidly gained popularity. Despite positive results in improving articular knee function and pain management , magnetiDespite these promising results, the exact mechanism of action of μFAT, and the way it may interact with the osteoarthritic microenvironment, is not fully understood and is supposed to rely on the release of soluble factors and EVs. The aim of this work was to characterize μFAT architecture in terms of structure and the most relevant stromal cell type composition, and compare with unprocessed lipoaspirate (LA) obtained from the same patients. For the first time, to our knowledge, we performed a deep characterization of both tissue and soluble factors in both products (μFAT and donor-matched LA) via proteomics and released EVs and embedded miRNAs via high-throughput qRT-PCR.® International, Milan, Italy). Both Lipogems 120TM and 240TM devices were used to highlight possible differences, in term of features of the final products. First, reduction of lipoaspirate clusters was performed by pushing the lipoaspirate through a filter into the device where five stainless steel beads facilitated the obtaining of an emulsion of oil, blood, and saline. The suspension was washed against density by means of a current of saline moved by gravity in the wasting bag. After washing, further adipose cluster reduction was performed through a second filter and µFAT was decanted by gravity in order to remove excessive saline solution. Adipose tissue was collected from 7 donors  during liposuction with Coleman’s technique . Part ofAdipose tissue samples were fixed in 10% neutral buffered formalin  for 24 h at RT, embedded in paraffin and sectioned at 4 μm. Hematoxylin and Eosin (H&E) staining was performed to evaluate the structure and morphology of the samples. The presence and localization of CD31, CD146 and CD90 markers were determined by immunohistochemistry staining. Briefly, sections were incubated with 1X Dewax and HIER Buffer H solution  at 60 °C overnight for antigen retrieval, permeabilized with 0.1% Triton X-100 in PBS for 20 min at RT, and incubated with blocking buffer 3% BSA in PBS for 1 h at RT. Samples were then incubated at 4 °C overnight with the following primary antibodies: rabbit monoclonal to CD31 , mouse monoclonal to CD90  and mouse monoclonal to CD146  antibodies. Sections were then washed with PBS buffer and incubated for 1h with anti-rabbit IgG, (H + L) raised in goat, biotinylated  or with anti-mouse IgG, (H + L) raised in horse, biotinylated  secondary antibodies. At the end of the immunohistochemistry staining, microscope images were acquired by means of an Olympus IX71  inverted microscope.All samples were incubated with collagenase type I 0.075% w/v  at 37 °C for 45 min in order to isolate the stromal vascular fraction (SVF) . WashingImmunophenotype analysis: Freshly isolated SVF (around 2 mL) was incubated for 10 min at RT with 10 volumes of Red Blood Cell Lysis Solution  to remove erythrocytes. After centrifugation, the pellet was washed once with 10 mL FACS buffer and eventually the pellet was suspended in 200 µL FACS buffer and divided into 2 aliquots. To identify the specific subpopulations , one aliSenescence analysis: For SVF obtained from LA, a 1:50 dilution was prepared in PBS. SVF  was split into 2 aliquots. One aliquot was left unstained and used as the negative control, while the other was stained with CD45-PE-Vio770  and CD235a-PE  antibodies for 30 min at 4 °C in the dark, following the manufacturer’s instructions. Then, both aliquots were washed with 1 mL Fluorescein di-β-D-galactopyranoside (FDG) buffer . The control aliquot was supplemented with 150 µL FDG buffer while the stained aliquot was divided into two 35 µL aliquots. One aliquot was supplemented with 35 µL FDG buffer as a negative control for FDG and the other with 35 µL 2 mM FDG, and both were incubated for 5 min at 37 °C in the dark. Then, both aliquots were supplemented with 80 µL ice-cold FDG buffer and all three samples  were analyzed using a flow cytometer  acquiring at least 30,000 events.3 cells/cm2  and ASCs were selected by plastic adherence. For adipogenic differentiation, a repeated pulsed protocol, consisting of 3 days in an adipogenic induction medium , followed by three days in a maintenance medium (control medium supplemented with 10 μg/mL insulin) was conducted, for a total of 21 days. Lipid vacuoles were visualized with Oil Red O . For osteogenic differentiation, osteogenic medium  was used for 21 days. Extracellular calcified matrix deposition was visualized with Alizarin Red S . For chondrogenic differentiation, chondrogenic medium  was used for 21 days. Chondrogenic proteins were visualized with Alcian blue .Following protocol for cell isolation as per 2. Then, the total volume was centrifuged at 376× g for 5 min at 4 °C and the liquid phase collected with a syringe. The liquid phase was filtered with a 100 µm cell strainer and centrifuged at 376× g for 5 min at 4 °C. The supernatant was sequentially centrifuged at 1000, 2000 and 4000 (twice)× g for 10 min at 4 °C. Eventually, the supernatant was filtered through a 1.2 µm filter and the cleared sample collected. The sample was further divided into three aliquots: 3 mL for EV retrieval and EV-miRNA detection, 2 mL for proteomic analysis, and the remaining for EV size distribution analysis. Aliquots were stored at −80 °C until use.LA or μFAT (around 6 mL each) were supplemented with DMEM in a 1:1 volume ratio, and incubated for 24 h at 37 °C in a humidified atmosphere at 5% COTissue samples were suspended in lysis buffer ) and sonicated. Supernatants were first concentrated with Amicon 5 kDa (Sigma-Aldrich) and mixed with 1% SDS and 5 mM DTT, prior to sonication.w/w) Trypsin Gold, MS grade  for 16 h at 37 °C.Protein concentration was determined by 2D Quant Kit . Following the FASP (Filter-Aided Sample Preparation) protocol , one-hunm/z) in the Orbitrap  and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan . Isolation window: 3 Da, unassigned charge states: rejected, charge state 1: rejected, charge states 2+, 3+, 4+: not rejected; dynamic exclusion enabled . Mass spectra were analyzed using MaxQuant software [The obtained peptides were subjected to LC-ESI-MS/MS shotgun analysis, performed on a Dionex UltiMate 3000 HPLC System with an Easy Spray PepMap RSLC C18 column  with the following gradient: 5% acetonitrile (ACN) in 0.1% formic acid for 10 min, 5–35% ACN in 0.1% formic acid for 79 min, 35–60% ACN in 0.1% formic for 40 min, 60–100% ACN for 1 min, 100% ACN for 10 min at a flow rate of 0.3 μL/min. The eluate was electrosprayed into an Orbitrap Tribrid Fusion (Thermo Fisher Scientific) through a nano-electrospray ion source (Thermo Fisher Scientific). The LTQ-Orbitrap was operated in positive mode in data-dependent acquisition mode to automatically alternate between a full scan (350–2000 software  (versionsoftware . Proteinsoftware . The req®) . The quantitative protein data were imported into IPA software to identify canonical pathways and diseases and biofunctions most strongly associated with the protein lists. The software works by experimental expression data on networks constructed from published interactions by giving a score (z-score), indicating up-regulation (z-score > 2) or down-regulation (z-score < 2).Bioinformatics analysis was carried out by Ingenuity Pathway Analysis (IPACleared samples (1:20 PBS diluted) were visualized by the Nanosight LM10-HS system , with five recordings of 30 s for each sample. NTA software was used to analyze the data and provided both the concentration measurements and the high-resolution particle size distribution profiles.® platform (Thermo Fisher Scientific). CDNAs were prepared by standard reverse transcription protocol, and preamplification performed with A and B independent kits, followed by real-time RT-PCR analysis with the QuantStudio™ 12 K Flex OpenArray® Platform (QS12KFlex) [RT values > 28 were considered as absence of amplification. The global mean of shared EV-miRNAs was used to normalize expression data [−ΔCRT.Secretome aliquots (3 mL) were diluted to 10 mL with triple 0.2 µm filtered PBS and centrifuged for 9 h at 100,000× g at 4 °C in a 70Ti rotor (Beckman). The pellet was dissolved in Trizol reagent (Thermo Fisher Scientific). MiRNeasy and RNeasy CleanUp Kits were sequentially used to isolate RNA enriched in small molecules (<200 nt) (Qiagen). Six pg of a non-human synthetic miRNA  were added before extraction to each sample as a spike-in to validate the technical variability during the RNA isolation procedure, during the following reactions and to equalize A and B panels of the OpenArray12KFlex) . The Expion data . EV-miRNAfter ultra-centrifugation, EV pellets were suspended in PBS . 5 μL were absorbed for 10 min at RT on formvar carbon-coated grids and excess liquid was removed with filter paper. Negative staining was done with 2% uranyl acetate aqueous suspension for 10 min and excess liquid was removed with filter paper. The grid was dried at RT. Samples were examined with a TALOS L120C transmission electron microscope  at 120 kV.https://biit.cs.ut.ee/clustvis/, accessed on 30 September 2021) [PCA and heat map plots were generated with ClustVis package (er 2021) . Clustert-test with post-hoc test Permutation based FDR < 0.05, conducted using Perseus software (version 1.6.1.3). Only proteins present and quantified in at least 80% of the technical and biological repeats were considered as positively identified in a sample and used for statistical analyses.Statistical Analyses were performed using Graphpad Prism v5.0 . The Shapiro-Wilk test was used to assess normal distribution of data. On the basis of these results, the comparison among groups was performed using one-way ANOVA for repeated measures, with Bonferroni’s post-test, or Friedman’s test, with Dunns’ post-test. For proteomics, Student TM and 240TM), and for ease of presentation, only data obtained with 120TM are herein presented.Due to identical results obtained with the two devices under study (Lipogems120+) embraced small vessels, without differences among µFAT and LA.Histological investigations showed a lower red blood cell presence in the μFAT compared to unprocessed LA, as emerged in the H and E staining . Blood v3/µL) 1.4 ± 0.6 vs. 0.4 ± 0.1, RBC (×103/µL) 177.1 ± 70.0 vs. 7.1 ± 2.9, PLT (×103/µL) 12.4 ± 6.4 vs. 3.0 ± 0.6, LA vs. µFAT mean ± SEM)), with µFAT/LA single ratios shown in p-value 0.040). These data were confirmed by flow cytometry analyses for leukocytes (CD45+) and erythrocytes (CD45−CD235a+), showing a 79%  and 76%  reduction, respectively. Eventually, viable cells reduced from 67% ± 6 to 49% ± 8 in µFAT with respect to unprocessed LA .Microfragmentation resulted in a loss of blood-related cells , ASCs (CD45−CD31−CD34+CD90+CD105−/LCD146−) and pericytes (CD45−CD31−CD34−CD90+CD105−CD146+) [p-values always ns) , while vascular endothelial cells and pericytes accounted for 7.9 ± 2.3% vs. 7.3 ± 0.9% and 1.6 ± 0.6% vs. 1.2 ± 0.4% (LA vs. µFAT mean ± SEM), respectively. Principal Component Analysis (PCA) associated with Hierarchical Clustering evaluated the effect of the processing on the ratios of the different stromal cell populations with respect to the original LA composition, and no distinct clusters emerged; suggesting that microfragmentation does not select for specific stromal populations (Both LA and μFAT CD45ytometry  for the −CD146+) . No signways ns) B. Adiposulations C,D. Isolulations .− population was similar before and after tissue processing, with very low values .Finally, the percentage of senescent cells in the CD45p ≤ 0.05) varied  are reported in In the tissues (µFAT and LA), around 870 proteins were considered valid for quantification, and 544 proteins significantly  varied . The difp ≤ 0.05) varied between µFAT and LA  are reported in In the supernatants, 287 proteins were considered valid for quantification, and 217 proteins significantly  differences were observed, with mean particle size of 107.7 nm ± 16.8 for LA and 106.5 nm ± 7.7 for μFAT. TEM confirmed NTA dimensional range in isolated EVs. In total, 376 and 381 miRNAs were detected in LA and µFAT samples (p ≤ 0.05) varied between µFAT and LA and 5 newly gained. A more accurate search was focused on the most abundant ones falling in the first quartile of detection  was applied. MiR-451a (μFAT vs. LA = 0.16 ± 0.07), highly abundant in EVs released by erythrocytes [Extracellular vesicles (EVs) released from LA and μFAT samples were analyzed by NTA . No sign samples , with 57etection , that alhrocytes , and miRhrocytes , emergedhrocytes ,33 was shrocytes . Among tp-value ≤ 0.1. Regarding macrophage polarization, in total, 11 miRNAs with M2 anti-inflammatory promoting features were detected , resulted in only µFAT-EVs in the first quartile.Eventually, identified miRNAs were compared with those recently described to have a protective role in OA  and M2 adetected , with 7 detected . AnalyziIn this report, a deep characterization of donor-matched LA and μFAT showed that microfragmentation does not alter adipose tissue architecture and stromal cell content, while it provides clearance of blood-derived elements. Moreover, proteomic analysis and EV-embedded miRNA quantification highlighted both the reduction of blood-derived, and pro-inflammatory, miRNAs, and an increase in cartilage-protective factors.+ endothelial cells. Nevertheless, the presence of blood-derived cells, such as erythrocytes drastically dropped, thereby reducing the pre-inflammatory actions attributed to these elements in the joint microenvironment. These data were further confirmed by cell count and flow cytometry data for mononuclear cells, platelets and erythrocytes, confirming that processing, through washing steps, allows for efficient removal of blood contaminants maintaining overall structure, including vessels, intact. Of note, flow cytometry data also confirmed the conserved presence of CD31+ endothelial cells between μFAT and LA. Of crucial importance for μFAT paracrine activity, both products had a similar content of active stromal cells, such as ASC and pericytes. Importantly, correlation analyses for the identified stromal cell types confirmed that, overall, the microfragmentation procedure does not significantly alter stromal cell ratios compared to LA. Taken together, these data clearly indicate that microfragmentation does not select for specific cell subpopulations and results in tissue with identical architectural and stromal composition, when compared to unprocessed LA.Microfragmentation was shown to produce smaller tissue pieces with respect to classical lipoaspiration using the Coleman technique, thus increasing the surface/volume ratio compared with larger tissue pieces . ConsistMicrofragmentation was able to produce changes, in terms of both tissue and released factors, if there was overall similar architecture and stromal cell composition. It clearly emerged that processing, as expected during tissue disruption, brought some stress and cell lysis as evidenced from reduction in cell viability and release of cytosolic proteins in the supernatant, as well as the detection of protein turnover-related factors and chaperones in the tissues. Moreover, proteomic data showed the attempt of microfragmented tissue to counteract tissue loosening by activating cell metabolism at different levels. Notably, acute phase factors significantly reduced in µFAT secretome. Acute phase proteins are a reaction to tissue injury, including the damaged joint, and involve inflammation . In partAdipose tissue EVs have been postulated as important mediators for the regenerative features of fat in the management of several diseases, including orthopaedic pathologies. In particular, miRNAs were the active players behind EVs’ action , and, thMMP3 and MMP13 metalloproteases [To substantiate the capacity of adipose tissue to counteract joint diseases, such as OA, miRNAs reported to have protective functions in OA  were sifroteases . miR-24-roteases . It shouroteases ,54. Therroteases . A similroteases , which sThese data, together with the soluble factors fingerprint, give molecular grounds for μFAT features reported in several models related to OA. In primary human chondrocytes, μFAT-induced cell proliferation, ECM synthesis and glycosaminoglycan level increased compared to LA . In rat Together with the relevant strength of comparing LA and µFAT obtained from the same patients and the use of cutting-edge technologies for secretome fingerprinting, we are aware that this study has limitations. First, donors of the study were subjected to liposuction for aesthetic procedures and not affected by musculoskeletal disorders, such as OA, as normally happens for patients undergoing orthobiologics- (including µFAT) based procedures. However, this decision was based on the need for sufficient µFAT allowing for all the analyses. Second, qRT-PCR-based miRNA detection relies on a reduced number of miRNAs that are rapidly increasing in their overall number. We preferred to use this solid technology, due to both the deep overall characterization and the bunch of OA-related data reported for the herein sifted molecules. Therefore, in the future, a larger number of miRNAs will have to be scored, based on available new literature. Third, the use of serum-free media for secretome collection is far from the OA environment, albeit, to date, culturing conditions for secretome analysis that avoid serum interference and recapitulating OA joints are still far from being defined. Again, future studies will be fundamental when OA-recapitulating conditions become clearly defined by clinical and scientific communities.In conclusion, microfragmented adipose tissue presents intact architecture and stromal cell composition with respect to LA, together with a reduced amount of blood elements that might be detrimental for the joint microenvironment. Also, μFAT showed a molecular signature enriched in anti-inflammatory and chondroprotective mediators. Taken together, cell and molecular data suggest that μFAT is a potent tool for management of joint degeneration, by addressing the main molecular pathways involved in this disorder. Whether μFAT is able to provide joint tissue regeneration, in addition to addressing pain and inflammation, has yet to be proven.


Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and characterization of a novel EmaSR TCS in A. baumannii. We constructed a Tn5-tagged mutagenesis library, from which an emaS sensor kinase gene and emaR response regulator gene were identified. We found that emaS/emaR single-mutants and double-mutants were unable to replicate in M9 medium with 1% ethanol as the single carbon source. Motility and biofilm formation were negatively affected in double-mutants, and transcriptomic analysis showed that mutation of emaSR dysregulated genes required for carbon metabolism. In addition, emaS/emaR single-mutants and double-mutants were unable to survive in acetic acid- and sodium acetate-containing medium, indicating that the EmaSR TCS is also important for acetate metabolism. Furthermore, virulence against Galleria mellonella was diminished in emaS/emaR single- and double-mutants. Taken together, these results show that this novel EmaSR TCS is involved in the regulation of A. baumannii ethanol metabolism and acetate metabolism, with important implications on motility, biofilm formation, and virulence if mutated. Further research on the underlying mechanisms is warranted.  Acinetobacter baumannii is an emerging Gram-negative nosocomial pathogen that is a rising cause of hospital-acquired infections  and 24 μL of 5N NaOH. Phos-tagTM containing 12% SDS-acrylamide resolving gel was prepared with 50 μL of 50 mM Phos-tagTM, 1.25 ml of 1.5M Tris-Cl (pH8.8), 2.12 ml of 30% acrylamide solution, 50 μL of 10 mM MnCl2, 50 μL of 10% SDS, 1. 25 mL of ddH2O, 25 μL of 10% ammonium persulfate (APS), and 5 μL of N,N,N′,N′-tetramethylethylenediamine (TEMED). A 6% staking gel was prepared with 1 mL of 0.5M Tris-Cl (pH 6.8), 800 μL of 30% acrylamide solution, 40 μL of 10% SDS, 2.12 mL of ddH2O, 40 μL of 10% APS and 4 μL of TEMED. Electrophoresis of prepared samples was conducted at 120 V at room temperature for 90 minutes [Wild-type and ΔS strains were cultured in LB broth with shaking at 37 °C overnight. Bacteria were subcultured in M9 medium containing 5 mM citrate with or without the presence of varying alcohols. Bacteria were collected after OD minutes . 600 was determined, with OD600 equalized to 1 for each bacterium. A 2 μL sample of each strain was dropped on 0.5% LB agar, and plates were incubated at 37 °C, with images taken at 12-h intervals for at least 96 h. Swimming areas were analyzed by ImageJ .Motility assays were performed as previously described, with minor modifications . Each st600 of 0.1. Bacteria were then treated with 10 mM H2O2 for 20 minutes and 4% NaCl, after which viable counts were determined by dropping 5 μL of cultured medium on LB agar plates 6 times, and then calculating colony-forming units (CFU) after colonies appeared on the plates [A single colony of each strain was inoculated in 3 mL of LB medium containing the corresponding antibiotics, and cultured for 12–16 hours at 37 °C with agitation. The overnight culture was refreshed with fresh medium to an OD600 of 0.1. Cultures were grown in 96-well microtiter plates for 10 hours, after which crystal violet was added to each well at a final concentration of 0.1%, and cultures were maintained at room temperature for 30 minutes, following which the crystal violet-containing medium was removed, and wells were subsequently washed twice with distilled water. The stained biofilms in each well were dissolved in 300 μL of 70% ethanol, and incubated for 15 minutes at room temperature before OD595 analysis.Biofilm formation assays were performed as previously described, with minor modifications . BacteriA. baumannii ATCC 19606 genome (GenBank accession numbers: SRX3312085 and SRX3312086) using SOAP2 software [RNA sequence reads generated from an Illumina HiSeq 2000  underwent selection, with reads containing adaptors, unknown sequences exceeding 10%, or low quality  removed. The clean reads were then mapped to the published software . Gene exsoftware . False d600 reached 0.3. Samples were then collected and mixed with 0.1 volume of fixing solution . After centrifugation by 17,000× g at 4 °C, the supernatant was discarded, and cell pellets were stored at −80 °C for RNA extraction. Cell pellets were thawed on ice and resuspended in 1 mL of NucleoZOL , then mixed thoroughly with 400 μL of diethyl pyrocarbonate (DEPC)-treated H2O, and incubated at room temperature for 15 minutes. The supernatant was recovered after centrifugation at 17,000× g at 4 °C for 20 minutes, then mixed with 5 μL of 100% 4-bromoanisole and incubated at room temperature for 10 minutes. Excess protein was then removed by centrifugation at 17,000× g at 4 °C for 20 minutes. The resulting RNA suspension was mixed with an equal volume of isopropanol for 15 minutes to induce RNA precipitation. The derived RNA pellet was washed twice with ice-cold 75% ethanol and resuspended in 30 μL of DEPC-treated H2O for analysis.Bacterial strains were cultured at 37 °C with agitation overnight, and then subcultured in 50 mL of M9 medium containing 5 mM citrate with or without 0.5% ethanol for at least 6 hours after ODA Nanodrop 2000C spectrophotometer  was used to determine RNA concentrations, and a total of 2 μg of RNA was used to prepare cDNA. The RT-PCR mixture contained 10× reaction buffer, 200 U of MMLV high performance reverse transcriptase , 100 mM of dithiothreitol (DTT), 2.5 mM dNTP, and 1 nM of hexamer. The reaction was conducted in a Biometra TADVANCED Thermal Cycler . Gene-specific primers used to determine the presence and expression levels of the respective genes are listed in A. baumannii ATCC 19606 wild-type and emaS, emaR, and emaSR mutant strains. All procedures were performed as previously described, with minor modifications [G. mellonella larvae with same total weight, and larvae were kept in petri dishes without food prior to infection. Overnight cultures of each strain were washed twice with PBS , then diluted in PBS. Larvae were subsequently infected with 5 × 106 CFU of each strain, with bacteria in 10-µl aliquots injected into the hemocoel of each larva via the last left proleg using a Hamilton syringe. Infected larvae were then incubated at 37 °C, and scored for survival  every 24 hours, as well as for melanization over 96 hours, based on a previously described scoring method [A virulence comparison was carried out with ications . We seleg method .